Precise breeding - backbone integration markers

ABSTRACT

The present invention relates to a new plant breeding process. The process improves the agronomic performance of crop plants by using genetic material that is also used in classical breeding. Instead of sexually recombining entire genomes at random, as is done in classical breeding, specific genetic elements are rearranged in vitro and inserted back into individual plant cells. Plants obtained through this new plant breeding process do not contain foreign nucleic acid but only contain nucleic acid from the plant species selected for transformation or plants that are sexually compatible with the selected plant species. Plants developed through this new plant breeding process are provided. In particular, potato plants displaying improved tuber storage and health characteristics are provided.

This application is a divisional application of U.S. application Ser.No. 10/369,324 filed on Feb. 20, 2003, and claims priority to U.S.provisional application Ser. Nos. 60/357,661 and 60/377,602, which areall incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for improving the nutritional,health, and agronomic characteristics of a plant by modifying specific,well-characterized DNA in the plant's genome. As opposed to classicalplant breeding, the inventive process does not introduce unknown orpotentially toxic genes into the plant genetic make-up. Furthermore, theinventive method, unlike conventional genetic engineering strategies,does not incorporate nucleic acids from foreign species, i.e., speciesthat are not inter-fertile with the plant to be modified by geneticengineering, into the plant genome. Plants developed through theinventive plant breeding process display improved agronomiccharacteristics. Particularly preferred plants of the present inventioninclude potatoes that exhibit improved health and tuber storagecharacteristics, and turfgrasses that exhibit improved disease anddrought tolerance.

BACKGROUND

The agronomic performance of plants has typically been improved byeither classical plant breeding or genetic engineering. Classicalbreeding typically results in the transfer of unknown nucleic acids fromone plant to another. Genetic engineering techniques introduce foreignnucleic acids into the plant genome, i.e., DNA that is not from a plantor that is not from a plant that is naturally interfertile with theplant to be modified by genetic engineering. For example, geneticengineering introduces non-plant nucleic acids into a plant genome. Bothclassical breeding and genetic engineering strategies create plantgenomes that contain undesirable and unwanted genetic material, and theresultant cross-bred or transgenic plants can exhibit unfavorabletraits. The inadequacies of both strategies can prove harmful to thetransgenic plants, as well as to the animals and humans who consume suchproducts.

Conventional Breeding Relies on the Transfer of Unknown DNA

Plant breeding typically relies on the random recombination of plantchromosomes to create varieties that have new and improvedcharacteristics. Thus, by screening large populations of progeny thatresult from plant crosses, breeders can identify those plants thatdisplay a desired trait, such as an increase in yield, improved vigor,enhanced resistance to diseases and insects, or greater ability tosurvive under drought conditions. However, classical breeding methodsare laborious and time-consuming, and new varieties typically displayonly relatively modest improvements.

Furthermore, classical plant breeding typically results in the transferof hundreds of unknown genes into a plant genome. It is likely that someof those transferred genes encode potentially harmful allergens, such aspatatin, lectins, chitinases, proteases, thaumatin-like proteins, lipidtransfer proteins, amylases, trypsin inhibitors, and seed storageproteins (Breiteneder et al., J Allergy Clin Immunol 106: 27-36).

Similarly, introgressed genes can be involved in the biosynthesis oftoxins including lathyrogens, hydrazines, glucosinolates and goitrogens,cumarins, saponins, alkaloids, glycoalkaloids, biogenic amines, enzymeinhibitors, such as lectins (haemagglutinins), trypsin inhibitors,chelating substances such as phytates and oxalates, ribotoxins,antimicrobial peptides, amino acids such asbeta-N-oxalylamino-L-alanine, atractyloside, oleandrine, taxol, andisoquinoline (Pokorny, Cas Lek Cesk 136: 267-70, 1997). The risk ofinadvertently introducing such poisons into human and animal foodsupplies is further increased through efforts to “untap” the geneticdiversity of wild crop relatives that have not been used before for foodconsumption (Hoisington et al., Proc Natl Acad Sci USA 96: 5937-43,1999).

Although classical plant breeding can easily introduce genes involved inundesirable anti-nutritional compounds into food crops and plants, itcannot easily remove them. For instance, it took about 15 years toreduce harmful phytate levels in corn and rice by inactivating Lpa genes(Raboy, J Nutr 132: 503S-505S, 2002). The long timeframe for realizingpositive results is not practical, especially since there is an urgentneed for methods that more effectively and efficiently improve thequality of food crops. One example of a gene that only recently wasfound to be associated with the synthesis of anti-nutritional compoundsis the polyphenol oxidase (PPO) gene, which oxidizes certain phenoliccompounds to produce mutagenic, carcinogenic and cytotoxic agents likephenoxyl radicals and quinoid derivatives (Kagan et al., Biochemistry33: 9651-60, 1994). The presence of multiple copies of this gene in thegenome of plants such as potato makes it particularly difficult toreduce PPO activity through breeding.

Even more time is needed for the removal of anti-nutritional compoundsif little or nothing is known about their genetic basis. For instance,no genes have been linked to the accumulation of high concentrations ofacrylamide, a potent neurotoxin and mutagen, in some potatoes that areheated to 160° C. or higher (Tareke et al., J Agric Food Chem. 50:4998-5006, 2002). It is therefore very difficult to efficiently developnew potato varieties that produce less acrylamide during processingusing conventional breeding. Thus, there is a need to grow potatoes andother carbohydrate-rich foods, such as wheat, with reduced levels ofsuch dangerous compounds, but without the use of unknown or foreignnucleic acids.

Other anti-nutritional compounds that can accumulate during processingand are difficult to minimize or eliminate through breeding include theMaillard-reaction productsN-Nitroso-N-(3-keto-1,2-butanediol)-3′-nitrotyramine (Wang et al., ArchToxicol 70: 10-5, 1995), and 5-hydroxymethyl-2-furfural (Janzowski etal., Food Chem Toxicol 38: 801-9, 2000). Additional Maillard reactionproducts that have not been well characterized are also known to displaymutagenic properties (Shibamoto, Prog Clin Biol Res 304: 359-76, 1989).

It can be equally difficult to rapidly increase levels of positivenutritional compounds in food crops due to the inherent imprecision ofconventional plant breeding. For instance, it would be desirable toincrease levels of “resistant starch” (Topping et al., Physiol Rev 81:1031-64, 2001) in a variety of crops. Such starch is ultimatelyresponsible for promoting immune responses, suppressing potentialpathogens, and reducing the incidence of diseases including colorectalcancer (Bird et al., Curr Issues Intest Microbiol 1: 25-37, 2000).However, the only available plants with increased levels of resistantstarch are low-yielding varieties like maize mutants “amylose extender”,“dull”, and “sugary-2.” Creation of new high resistant starch sources,such as potato, would enable broader dietary incorporation of thishealth-promoting component.

The inability to safely manipulate the genotypes of plants often leadsto the use of external chemicals to induce a desired phenotype. Despitenumerous breeding programs to delay tuber sprouting, for example, nopotato varieties are available commercially that can be stored formonths without treatment with sprout inhibitors. The latter, such asisopropyl-N-chlorophenyl-carbamate (CIPC), is linked to acute toxicityand tumor development, and can be present in processed potato foods atconcentrations between 1 mg/kg and 5 mg/kg.

Genetic Engineering Relies on the Transfer of Foreign DNA

Genetic engineering can be used to modify, produce, or remove certaintraits from plants. While there has been limited progress in improvingthe nutritional value and health characteristics of plants, mostimprovements target plant traits that promote ease of crop cultivation.Thus, certain plants are resistant to the glyphosate herbicide becausethey contain the bacterial gene 5-enolpyruvylshikimate-3-phosphatesynthase (Padgette et al., Arch Biochem Biophys. 258: 564-73, 1987).Similarly, genetic engineering has produced insect-, viral-, andfungal-resistant plant varieties (Shah et al., Trends in Biotechnology13: 362-368, 1995; Gao et al., Nat Biotechnol. 18: 1307-10, 2000; Osuskyet al., Nat Biotechnol. 18: 1162-6, 2000), but few with enhancednutrition or health benefits.

According to standard, well-known techniques, genetic “expressioncassettes,” comprising genes and regulatory elements, are insertedwithin the borders of Agrobacterium-isolated transfer DNAs (“T-DNAs”)and integrated into plant genomes. Thus, Agrobacterium-mediated transferof T-DNA material typically comprises the following standard procedures:(1) in vitro recombination of genetic elements, at least one of which isof foreign origin, to produce an expression cassette for selection oftransformation, (2) insertion of this expression cassette, oftentogether with at least one other expression cassette containing foreignDNA, into a T-DNA region of a binary vector, which usually consists ofseveral hundreds of basepairs of Agrobacterium DNA flanked by T-DNAborder sequences, (3) transfer of the sequences located between theT-DNA borders, often accompanied with some or all of the additionalbinary vector sequences from Agrobacterium to the plant cell, and (4)selection of stably transformed plant cells. See, e.g., U.S. Pat. Nos.4,658,082, 6,051,757, 6,258,999, 5,453,367, 5,767,368, 6,403,865,5,629,183, 5,464,763, 6,201,169, 5,990,387, 4,693,976, 5,886,244,5,221,623, 5,736,369, 4,940,838, 6,153,812, 6,100,447, 6,140,553,6,051,757, 5,731,179, 5,149,645 and EP 0 120,516, EP 0 257,472, EP 0561,082, 1,009,842A1, 0 853,675A1, 0 486,233B1, 0 554,273A1, 0270,822A1, 0 174,166A1, and WO 01/25459.

Thus, genetic engineering methods rely on the introduction of foreignnucleic acids into the food supply. Those techniques transfer complexfusions of a few to more than 20 genetic elements isolated from viruses,bacteria, and plants, that are not indigenous to the transformed plantspecies. Such foreign elements include regulatory elements such aspromoters and terminators, and genes that are involved in the expressionof a new trait or function as markers to identify or select fortransformation events. Despite the testing of foods containing foreignDNA for safety prior to regulatory approval, many consumers areconcerned about the long-term effects of eating foods that expressforeign proteins, which are produced by genes obtained from other,non-plant species.

One commonly used regulatory element is the 35S “super” promoter ofcauliflower mosaic virus (CaMV), which is typically used in plantengineering to induce high levels of expression of transgenes to whichit is directly linked. However, the 35S promoter also can enhance theexpression of native genes in its vicinity (Weigel et al., PlantPhysiol., 122: 1003-13, 2000). Such promoters may thus induceunpredictable alterations in the expression of endogenous genes,possibly resulting in undesirable effects such as increased alkaloidproduction. Preferred “strong” promoters are generally those isolatedfrom viruses, such as rice tungro bacilliform virus, maize streak virus,cassava vein virus, mirabilis virus, peanut chlorotic streakcaulimovirus, figwort mosaic virus and chlorella virus. Other frequentlyused promoters are cloned from bacterial species and include thepromoters of the nopaline synthase and octopine synthase gene.

To obtain appropriate termination of gene translation, terminatorsequences are fused to the 3′-end of transgenes and include geneticelements from the nopaline synthase and octopine synthase genes fromAgrobacterium. Other genetic elements may be used to further enhancegene expression or target the expressed protein to certain cellcompartments. These elements include introns to boost transgeneexpression and signal peptide sequences to target the foreign gene tocertain cellular compartments, often derived from foreign plant species.

Certain genes involved in expression of a new trait are most frequentlyderived from foreign sources. If native genes are used, they are ofteninverted to silence the expression of that gene in transgenic plants andco-transformed with foreign DNA such as a selectable marker. The maindisadvantage of this “antisense” technology is that the inverted DNAusually contains new and uncharacterized open reading frames insertedbetween a promoter and terminator. Thus, potato plants that weregenetically modified with antisense constructs derived from the starchrelated gene R1 (Kossmann et al., U.S. Pat. No. 6,207,880), the L- andH-type glucan phosphorylase genes (Kawchuk et al., U.S. Pat. No.5,998,701, 1999), the polyphenol oxidase gene (Steffens, U.S. Pat. No.6,160,204, 2000), and genes for starch branching enzymes I and II(Schwall et al., Nature Biotechnology 18: 551-554, 2000) all potentiallyexpress new peptides consisting of at least 50 amino acids (Table 1).These new peptides may interfere with plant development and/or reducethe nutritional value of potato, and are therefore undesirable.

Conventional marker genes are incorporated into genetic constructs andused to select for transformation events. They confer either antibioticor herbicide resistance (U.S. Pat. No. 6,174,724), a metabolic advantage(U.S. Pat. No. 5,767,378), or a morphologically abnormal phenotype (U.S.Pat. No. 5,965,791) to the transformed plant. Such markers are typicallyderived from bacterial sources.

Furthermore, because of the infidelity of T-DNA transfer, about 75% oftransformation events in plants such as tomato, tobacco, and potatocontain plasmid “backbone” sequences in addition to the T-DNA (Kononovet al., Plant J 11: 945-57, 1997). The presence of such backbonesequences is undesirable because they are foreign and typically containorigins of replication and antibiotic resistance gene markers.

There do exist various methods for removing elements like foreign markergenes, but few are easily applicable to plant genetic engineering.According to one such method, the marker gene and desired gene ornucleotide sequence are placed on different vectors. The infection ofplants with either a single Agrobacterium strain carrying both vectors(U.S. Pat. No. 6,265,638) or two Agrobacterium strains each of whichcarries one of the vectors can occasionally result in unlinkedintegration events, which may be separated genetically throughoutbreeding. The main disadvantage of this method is that the geneticseparation of loci can be very laborious and time-consuming, especiallyif T-DNA integration events are linked. Furthermore, this method is notwidely applicable in apomictic plants, which reproduce asexually, suchas Kentucky bluegrass, or vegetatively propagated crops such as potato,which cannot be readily bred due to inbreeding depression, high levelsof heterozygosity, and low fertility levels.

Another method for removing foreign genetic elements relies on insertingthe foreign gene, like the selectable marker, into a transposableelement. The modified transposable element may then be spliced out fromthe genome at low frequencies. Traditional crosses with untransformedplants must then be performed to separate the transposed element fromthe host (U.S. Pat. No. 5,482,852). As described for the previousmethod, this alternative method cannot be used for vegetativelypropagated or apomictic plant systems.

A third method of removing a marker gene uses the Cre/lox site-specificrecombination system of bacteriophage P1 (Dale & Ow, Proc. Natl. Acad.Sci. USA, 88: 10558-62, 1991). Insertion of a marker gene together withthe Cre recombinase gene and a chimeric gene involved in induction ofCre (both with their own promoters and terminators) between two loxsites leads to excision of the region delineated by the lox sites duringthe regeneration process (Zuo et al., Nat. Biotechnol., 19: 157-61,2001). This complicated process is inefficient and not reliable, and maycause genome instability.

Recent studies report that some plant genes themselves may be used astransformation markers. Examples of such plant markers include Pga22(Zuo et al., Curr Opin Biotechnol. 13: 173-80, 2002), Cki1 (Kakimoto,Science 274: 982-985, 1996) and Esr1 (Banno et al., Plant Cell 13:2609-18, 2001). All of the genes, however, trigger cytokinin responses,which confer an undesirable phenotype to the transformed plant.Furthermore, such plant markers would still need to be removed upontransformation by any of the methods described above.

Alternative methods to transform plants are also based on the in vitrorecombination of foreign genetic elements, and rely on bacterial plasmidsequences for maintenance in E. coli, parts of which are co-integratedduring the transformation process. Examples of such methods to transformplants with foreign DNA are described in U.S. Pat. Nos. 5,591,616,6,051,757, 4,945,050, 6,143,949, 4,743,548, 5,302,523, and 5,284,253.

Marker-free transgenic plants may also be obtained by omitting anyselection procedures prior to regeneration. A disadvantage of thismethod is that most events generated through this method will representuntransformed or chimeric plants because they will usually not bederived from single transformed plant cells. It is extremely difficultand laborious to use a marker-free procedure for the identification oftransgenic plants that contain the same DNA insertion(s) in all theircells.

Thus, there is a very important need to improve plants beyond that whichcan be accomplished through the classical breeding crosses andconventional genetic engineering techniques, and which does not rely onthe insertion of unknown or foreign nucleic acid into a plant genome.Accordingly, the present invention provides methods and compositions forprecisely modifying a plant's own genetic material. Thus, the inventive“precise breeding” strategy does not induce undesirable phenotypes anddoes not introduce unknown or foreign nucleic acid into a plant genome.

SUMMARY

The present invention provides methods of genetically enhancing thenutritional value and agronomic performance of a plant without thepermanent or stable incorporation of either unknown or foreign DNA intothe genome of that plant. According to the methods of the presentinvention, specific, well-characterized nucleic acids, gene elements,and genes are isolated from a desired plant species or from a plantspecies that is sexually compatible with the desired plant, modified,and then reinserted back into the genome of the desired plant species.The modification may entail mutating the isolated nucleic acid sequence,deleting parts of the isolated nucleic acid, or simply joining theisolated nucleic acid to another polynucleotide, such as subcloning theisolated nucleic acid into a plasmid vector.

Accordingly, transgenic plants produced by the inventive methodology donot possess genomes that comprise any foreign species' nucleic acids.Thus, the methods of the present invention produces a transgenic plantwhose genome does not comprise a non-plant species promoter, does notcomprise a non-plant species terminator, does not comprise a non-plantspecies 5′-untranslated region, does not comprise a non-plant species3′-untranslated region, does not comprise a non-plant species markergene, does not comprise a non-plant species regulatory element, does notcomprise a non-plant species gene, and does not comprise any otherpolynucleotide that is obtained from a non-plant species genome.

Thus, the present invention provides a method for producing a stabletransgenic plant that exhibits a modified phenotype that is notexhibited by the non-transformed plant, comprising (a) transformingplant cells with a desired polynucleotide; (b) growing plants from thetransformed cells; and (c) selecting a plant stably transformed withsaid desired polynucleotide which exhibits a new phenotype that is notexhibited by plants grown from the corresponding non-transformed plantcells. Preferably, the desired polynucleotide consists essentially of(i) nucleic acid sequences that are isolated from and/or native to thegenome of the plant cells, or to other plants of the same species, orare isolated from and/or native to the genome of a plant species that issexually compatible with the plant from which the plant cells wereisolated; and (ii) at least one DNA sequence that is a border-likesequence that has a sequence that is native to the genome of said plantcells or is native to the genome of plant cells of the same species, oris native to a plant that is sexually compatible with the plant fromwhich the plant cells were isolated, and wherein the border-likesequence is capable of stably integrating the desired polynucleotideinto the genome of said plant cells.

A preferred method of the present invention entails producing atransgenic plant that exhibits a modified phenotype that is notexhibited by the non-transformed plant, comprising (a) infectingexplants with Agrobacterium carrying (i) a “P-DNA” vector, whichcontains a desired polynucleotide that is native to the transgenicplant, and (ii) a “LifeSupport” vector that contains an expressioncassette containing a selectable marker gene; (b) selecting fortransient expression of the selectable marker gene, preferably for 1 -10days, for 3-7 days, or for 4-5 days; (c) transferring explants toregeneration media to allow shoot formation; (d) screening populationsof shoots to determine which comprise at least one copy of the desiredpolynucleotide in their genomes and, of those, which shoots do notcontain any foreign nucleic acids, such as the selectable marker gene,in their genomes; and (e) allowing shoots which contain the desiredpolynucleotide in their genomes but not any marker gene DNA, to developinto whole plants, wherein the resultant whole plants exhibit a modifiedphenotype that is not exhibited by plants grown from non-transformedplant cells of the same species.

According to such a method, the desired polynucleotide (i) consistsessentially of only elements that are isolated from and/or native to thegenome of the plant cell species or sexually compatible species thereof;(ii) comprises at least one border element that has a sequence that isisolated from, or native to, the genome of the plant cell species orsexually compatible species thereof, and is capable of stablyintegrating the desired polynucleotide into the genome of a plant cellexposed to the vector; and (iii) is stably integrated into the genome ofthe transformed plant; wherein the method does not integrate non-plantspecies or foreign DNA into the genome of the transformed plant.

Furthermore, any selectable marker gene may be used as an indicator ofsuccessful transformation. For instance, a “neomycin phosphotransferase”marker gene, or an “hpt” marker gene may be used to confer resistance tothe aminoglycoside antibiotics, kanamycin and hygromycin respectively.Other marker genes include the “bar” marker gene, which confersresistance to herbicide phosphinothricin; the “DHFR” marker gene, whichconfers resistance to methotrexate; and the “ESPS” marker gene, whichconfers resistance to Round-up herbicide. It is well known in the arthow to follow expression of such marker genes to determine whether ornot the marker gene has been stably expressed into the genome of atransformed plant cell. Accordingly, the skilled artisan knows how tofollow expression of the marker gene to determine that the marker geneis only transiently expressed in the transformed plant cell.

In another aspect of the invention, there is provided a method of makinga stably transformed plant comprising the steps of: (1) identifying atarget gene; (2) isolating a leader or trailer DNA sequence associatedwith said target gene; (3) optionally modifying said isolated leader ortrailer DNA; (4) operably linking said leader or trailer DNA to nativeregulatory elements to form an expression cassette; (5) inserting saidexpression cassette into a P-DNA that is located on a binary vector,wherein the binary vector also carries an operable cytokinin gene suchthat the inadvertent insertion of additional binary vector sequences,which are of foreign origin, are detected by expression of the cytokiningene; (6) introducing the modified binary vector into Agrobacterium; (7)stably integrating the rearranged native DNA into the genomes of plantcells using LifeSupport-mediated transformation; (8) regenerating plantcells that contain the rearranged native DNA; (9) discarding plants thatdisplay a cytokinin-overproducing phenotype and do not fully regenerate;and (10) maintaining for further analysis the desirable plants that areindistinguishable from untransformed plants.

In another aspect of the instant invention, a method of modifying theexpression of a trait in a selected plant species is provided. In oneembodiment, the method comprises (1) identifying the trait to bemodified; (2) constructing a recombinant DNA molecule consistingessentially of genetic elements isolated from, or native to, theselected plant species, wherein the recombinant DNA molecule, whenintegrated into the genome of the selected plant species, modifies theexpression of the trait in the transformed plant species; (3) stablyintegrating the recombinant DNA molecule into cells of the selectedplant species using LifeSupport-mediated transformation; and (4)identifying transformed plants exhibiting modified expression of thetrait.

In a preferred embodiment, polynucleotide that is native to a desiredplant is inserted into the desired plant's genome by infecting explantswith two different Agrobacterium strains. A first Agrobacterium strainis capable of transferring the native DNA from P-DNA vectors to plantcells; a second strain can transfer a T-DNA carrying an expressioncassette for a selectable marker gene to plant cells. Examples of thelatter vector include the so-called, “LifeSupport” vectors describedherein. By preferably selecting plants that transiently express themarker gene for 1-10 days, for 3-7 days, or for 4-5 days, andsubsequently transferring explants to regeneration media, a populationof events is obtained, part of which represents plants that contain atleast one copy of the polynucleotide, but which lack any copies of theT-DNA or marker gene.

In another embodiment, a single Agrobacterium strain is used thatcarries both a P-DNA vector, which houses the desired, native gene ofinterest or polynucleotide between P-DNA border-like sequences, and aLifeSupport vector, which contains a marker gene. The marker gene may,or may not, be inserted between P-DNA border-like sequences, T-DNAborder sequences, or other T-DNA-like border sequences.

Thus, in another preferred embodiment, the P-DNA vector contains atleast two expression cassettes, one of which comprises a nativescreenable or selectable marker gene driven by a native promoter andfollowed by a native terminator.

By preferably selecting for at least 2 days and more preferably for atleast 5 days for native marker gene expression and subsequentlytransferring explants to regeneration media, a population of events isobtained that represent plants containing at least one copy of theintroduced DNA stably integrated into their genomes. In preferredembodiments, the plant-derived marker gene encodes a mutant5-enolpyruvul-3-phosphoshikimic acid synthase or tryptophandecarboxylase. In a more preferred embodiment, the selectable markerencodes for salt tolerance. In a most preferred embodiment, the salttolerance gene has the nucleotide sequence shown in SEQ ID 35 and isused to select for transformation events in potato.

In yet another embodiment, the modified expression of the trait ischaracterized by an increase in expression, a decrease in expression, orin undetectable expression.

In another aspect of the instant invention, a plant made by the methodof (1) identifying the trait to be modified; (2) constructing arecombinant DNA molecule consisting essentially of genetic elementsisolated from the selected plant species, wherein the recombinant DNAmolecule when integrated into the genome of the selected plant speciesmodifies the expression of the trait in the transformed plant species;(3) stably integrating the recombinant DNA molecule into cells of theselected plant species through LifeSupport-mediated transformation; and(4) identifying transformed plants exhibiting modified expression of thetrait, is provided.

In a further aspect, a method of modifying expression of a trait in aselected plant species is provided. This method comprises (1)identifying the trait to be modified; (2) constructing a recombinant DNAmolecule consisting essentially of (a) genetic elements isolated fromthe selected plant species, wherein the genetic elements when integratedinto the genome of the selected plant species modifies the expression ofthe trait in the transformed plant species; and (b) a selectable markergene that is isolated from the same plant species; (3) stablyintegrating the recombinant DNA molecule into cells of the selectedplant species through LifeSupport-mediated transformation; (4) detectingthe selectable marker gene; and (5) identifying transformed plantsexhibiting modified expression of the trait.

In yet one other aspect, a plant exhibiting a modified expression of atrait is provided. In one embodiment, the plant has stably integratedinto its genome a recombinant DNA molecule consisting essentially ofgenetic elements isolated from a plant of the same species, or from aplant that is sexually compatible with that species, wherein therecombinant DNA molecule modifies the expression of the trait.

In another aspect of the present invention, an isolated nucleotidesequence referred to as “plant-DNA” (“P-DNA”) is provided. In apreferred embodiment, the P-DNA itself lacks any genes or parts thereofand is delineated by terminal, T-DNA “border-like” sequences that shareat least 50%, at least 75%, at least 90% or at least 95% sequenceidentity with the nucleotide sequence of the T-DNA borders of anyvirulent Agrobacterium strain, and which support an efficient transferof the entire P-DNA from Agrobacterium to plant cells.

In a preferred embodiment a “border-like” sequence promotes andfacilitates the integration of a polynucleotide to which it is linked.In another preferred embodiment, each terminal sequence of the modifiedP-DNA is between 5-100 bp in length, 10-80 bp in length, 15-75 bp inlength, 15-60 bp in length, 15-50 bp in length, 15-40 bp in length,15-30 bp in length, 16-30 bp in length, 20-30 bp in length, 21-30 bp inlength, 22-30 bp in length, 23-30 bp in length, 24-30 bp in length,25-30 bp in length, or 26-30 bp in length. More preferably, theborder-like sequence is between 20 and 28 nucleotides in length.

In a preferred embodiment, the P-DNA left and right border sequences ofthe present invention are isolated from and/or are native to the genomeof a plant that is to be modified and are not identical in nucleotidesequence to any known Agrobacterium-derived T-DNA border sequence. Thus,in one embodiment, a P-DNA border sequence may possess 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotidesthat are different from a T-DNA border sequence from an Agrobacteriumspecies, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes.Alternatively, in another embodiment, a P-DNA border, or a border-likesequence of the present invention has at least 95%, at least 90%, atleast 80%, at least 75%, at least 70%, at least 60% or at least 50%sequence identity with a T-DNA border sequence from an Agrobacteriumspecies, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes.More preferably, a native plant P-DNA border sequence that sharesgreater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%,76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,62%, 61%, or 60% nucleotide sequence identity with an AgrobacteriumT-DNA border sequence.

In another preferred embodiment, a border-like sequence can be isolatedfrom a plant genome and then modified or mutated to change theefficiency by which they are capable of integrating a nucleotidesequence into another nucleotide sequence. In another embodiment, otherpolynucleotide sequences may be added to or incorporated within aborder-like sequence of the present invention. Thus, in yet anotherembodiment, a P-DNA left border or a P-DNA right border may be modifiedso as to possess 5′- and 3′-multiple cloning sites, or additionalrestriction sites. In a further embodiment, a P-DNA border sequence maybe modified to increase the likelihood that backbone DNA from theaccompanying vector is not integrated into the plant genome.

In an even more preferred embodiment, the P-DNAs are isolated from anyplant by using degenerate primers in a polymerase chain reaction. In onepreferred embodiment, the P-DNA is derived from potato, is delineated by25-bp termini with 80 and 88% identity to conventional T-DNA borders,respectively, and has the nucleotide sequence shown in SEQ ID NO. 1. Inanother most preferred embodiment, the P-DNA is derived from wheat, isdelineated by 25-bp termini with 72% and 92% identity with conventionalT-DNA borders, respectively, and contains the nucleotide sequence shownin SEQ ID NO. 34.

Such a P-DNA may be modified so as to comprise other polynucleotidespositioned between the border-like sequences. In a preferred embodiment,the modified P-DNA consists essentially of, in the 5′- to 3′-direction,a first border-like sequence that promotes DNA transfer, a promoter, adesired polynucleotide that is operably linked to the promoter, aterminator and a second border-like sequence that also promotes DNAtransfer. In one other embodiment, the desired polynucleotide representsone or several copies of a leader, a trailer or a gene in sense and/orantisense orientations. In a more preferred embodiment, the modifiedP-DNA contains expression cassettes for both a mutant PPO gene and aninvertase inhibitor gene.

Thus, in a preferred embodiment, the desired polynucleotide comprises asense and antisense sequence of a leader sequence. In a more preferredembodiment, the leader sequence is associated with a gene that isendogenous to a cell of the selected plant species. In yet a morepreferred embodiment, the leader is associated with a gene that isselected from the group consisting of a PPO gene, an R1 gene, a type Lor H alpha glucan phosphorylase gene, an UDP glucose glucosyltransferasegene, a HOS1 gene, a S-adenosylhomocysteine hydrolase gene, a class IIcinnamate 4-hydroxylase gene, a cinnamoyl-coenzyme A reductase gene, acinnamoyl alcohol dehydrogenase gene, a caffeoyl coenzyme AO-methyltransferase gene, an actin depolymerizing factor gene, a Nin88gene, a Lol p 5 gene, an allergen gene, a P450 hydroxylase gene, anADP-glucose pyrophosphorylase gene, a proline dehydrogenase gene, anendo-1,4-beta-glucanase gene, a zeaxanthin epoxidase gene, and a1-aminocyclopropane-1-carboxylate synthase gene.

In yet another preferred embodiment, the desired polynucleotide sequencecomprises a sense and antisense sequence of a trailer sequence. In apreferred embodiment, the trailer sequence is associated with a geneselected from the group consisting of a PPO gene, an R1 gene, a type Lor H alpha glucan phosphorylase gene, an UDP glucose glucosyltransferasegene, a HOS1 gene, a S-adenosylhomocysteine hydrolase gene, a class IIcinnamate 4-hydroxylase gene, a cinnamoyl-coenzyme A reductase gene, acinnamoyl alcohol dehydrogenase gene, a caffeoyl coenzyme AO-methyltransferase gene, an actin depolymerizing factor gene, a Nin88gene, a Lol p 5 gene, an allergen gene, a P450 hydroxylase gene, anADP-glucose pyrophosphorylase gene, a proline dehydrogenase gene, anendo-1,4-beta-glucanase gene, a zeaxanthin epoxidase gene, and a1-aminocyclopropane-1-carboxylate synthase gene.

In a preferred embodiment, the desired polynucleotide, such as a gene,is isolated from, and/or is native to the plant that is to betransformed. In another preferred embodiment, the desired polynucleotideis modified or mutated. In one embodiment, a mutation to the isolatedpolynucleotide may render the desired nucleotide greater than or equalto 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%,85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%,71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% dissimilarto its unmutated form.

In a preferred embodiment of the present invention, the promoter of anexpression cassette located within a P-DNA is a constitutive promoter.In a more preferred embodiment the constitutive promoter is the promoterof the Ubiquitin-3 gene of potato. In an even more preferred embodimentthe constitutive promoter is the promoter of the Ubiquitin-7 gene ofpotato.

In another embodiment, the promoter of an expression cassette locatedwithin a P-DNA is a regulatable promoter. In a more preferredembodiment, the regulatable promoter is sensitive to temperature. In aneven more preferred embodiment, the regulatable promoter is a ci21Apromoter or a C17 promoter, each isolated from potato (Schneider et al.,Plant Physiol. 113: 335-45, 1997; Kirch et al., Plant Mol Biol 33:897-909, 1997).

In another embodiment, the promoter of an expression cassette locatedwithin a P-DNA can be regulated in a temporal fashion. In a preferredembodiment, the promoter is an rbcS promoter (Ueda et al., Plant Cell 1:217-27, 1989).

In yet another embodiment, the promoter of an expression cassettelocated within a P-DNA is regulated by any one of abscisic acid,wounding, methyl jasmonate or gibberellic acid. In a further embodiment,this promoter is a promoter selected from either a Rab 16A genepromoter, an α-amylase gene promoter or a pin2 gene promoter.

In another embodiment, the promoter of an expression cassette locatedwithin a P-DNA is a tissue-specific promoter. In a particularlypreferred embodiment, this promoter is a GBSS promoter isolated from S.tuberosum.

In one embodiment, the present invention provides a P-DNA vector that iscapable of replication in both E. coli and Agrobacterium, and containseither a P-DNA or a modified P-DNA. In a preferred embodiment, thisvector also contains an expression cassette for a cytokinin gene in itsbackbone to enable the selection against backbone integration events.

In another preferred embodiment, the desired nucleotide sequence furthercomprises a spacer element. In a more preferred embodiment, the spacerelement is a Ubi intron sequence or a GBSS spacer sequence.

In another preferred embodiment, the desired nucleotide sequencecomprises a mutated native gene encoding a functionally inactiveprotein, which reduces the overall activity of that protein if expressedin transgenic plants. In yet a more preferred embodiment, this mutatedgene encodes a functionally inactive polyphenol oxidase lacking a copperbinding domain.

In another preferred embodiment, the desired nucleotide sequencecomprises a native gene encoding a functionally active protein. In yet amore preferred embodiment, this gene encodes for a protein with homologyto the tobacco vacuolar invertase inhibitor.

In another embodiment, the terminator of an expression cassette locatedwithin a P-DNA is a Ubi3 terminator sequence or a 3′-untranslated regionof a gene of a selected plant species.

In another aspect of the instant invention, a method for modifying atarget plant cell is provided. In one embodiment, the method comprises:(1) inserting a modified P-DNA into the genome of at least one cell inthe target plant cell using LifeSupport-mediated transformation; and (2)observing if there is a phenotypic change in the target plant cell;wherein the promoter in the modified P-DNA transcribes the sense and/orantisense untranslated sequences associated with a native gene to reduceexpression of that native gene, thereby modifying the target plant cell.In another preferred embodiment, the promoter in the modified P-DNAtranscribes a gene to overexpress that gene in the target plant cell.

In yet another aspect, there is provided a method of making a transgenicplant cell of a selected plant species that contains a modified P-DNA.The method comprises co-transfecting a plant cell of the selected plantspecies with a P-DNA vector and a LifeSupport vector that comprises amarker gene flanked by a T-DNA left border and a T-DNA right border anda mutant virD2 gene inserted into the vector backbone, and selecting fora plant cell that transiently expresses the marker gene, and isolating aplant cell that contains the modified P-DNA integrated into its genomebut does not contain any nucleotides from the LifeSupport vector. In apreferred embodiment, the marker gene confers resistance to kanamycin.In a most preferred embodiment the yeast ADH terminator follows thekanamycin resistance gene.

In a preferred embodiment, the plant cell of the selected plant speciestargeted for transformation is in culture. In another preferredembodiment, the plant cell of the selected plant species targeted fortransformation is within a plant.

The present invention also provides a plant of the selected species thatcomprises at least one cell with a genome that contains a modifiedP-DNA. In a preferred embodiment, the modified P-DNA consistsessentially of, in the 5′- to 3′-direction, a first terminus thatfunctions like a T-DNA border followed by P-DNA sequences, a promoter, adesired nucleotide sequence operably linked to both a promoter, aterminator and additional P-DNA sequences delineated by a secondterminus. In another embodiment, the desired polynucleotide representsone or several copies of a leader, a trailer and a gene in the senseand/or antisense orientation.

In another embodiment, a plant that comprises at least one cell with agenome that contains a modified P-DNA is envisioned.

In another aspect of the invention, a method for reducing the expressionof a gene in a selected plant species is provided. The method comprisesthe LifeSupport-mediated transformation of a plant cell from a selectedplant species with a P-DNA vector, wherein the modified P-DNA of thisvector is stably integrated into the genome of the plant cell. Inanother aspect of the invention, the modified P-DNA comprises a desiredpolynucleotide that reduces expression of an endogenous gene from theselected plant species.

In another aspect of the instant invention, a gene native to theselected plant species may be mutated and reintroduced into the plantusing the inventive methods. Preferably, the mutated gene, for instancea mutated PPO gene, is integrated into the plant cell genome using aP-DNA vector.

The present invention also provides a method for reducing theundesirable expression of the polyphenol oxidase gene in a selectedplant species. In a preferred embodiment, the method comprisesintegrating into a genome of a selected plant species a modified P-DNAcomprised only of nucleotide sequences isolated from the selected plantspecies or from a plant that is sexually compatible with the selectedplant species, consisting essentially of, in the 5′- to 3′-direction, afirst P-DNA terminus that functions like a T-DNA border followed byflanking P-DNA sequences; a promoter; a desired nucleotide which is asense-oriented trailer nucleotide sequence associated with a specificPPO gene; an antisense-oriented sequence of the trailer nucleotidesequence from the specific PPO gene; a termination sequence, andadditional P-DNA sequences delineated by a second terminus thatfunctions like a T-DNA border, wherein the promoter produces adouble-stranded RNA molecule that reduces the expression of the specificPPO gene, thereby reducing black spot bruising in specific tissues ofthe plant. In another embodiment, the sense- and antisense-orientednucleotide sequences from the leader nucleotide sequences are obtainedfrom the 5′-untranslated region preceding the specific PPO gene. In afurther embodiment, the sense- and antisense-oriented leader or trailersequence associated with the PPO gene may be separated by anotherpolynucleotide sequence, referred to herein, as either an intron or a“spacer.” In a preferred embodiment, the leader or trailer sequence isassociated with a potato PPO gene. In a more preferred embodiment, theleader or trailer sequence is associated with a potato PPO gene that isexpressed in potato tubers. In a most preferred embodiment, the leaderor trailer sequence is associated with a potato PPO gene that isexpressed in all parts of the potato tuber except for the epidermis.

The present invention also provides a method for reducing acrylamideproduction, sprout-induction during storage, phosphate accumulation,and/or cold-induced sweetening in tubers of a selected plant species.

In a preferred embodiment, the method comprises the LifeSupport-mediatedtransformation of a selected plant species with a modified P-DNAcomprised only of nucleotide sequences isolated from the selected plantspecies, or from plants that are sexually compatible with the selectedplant species, consisting essentially of, in the 5′- to 3′-direction, afirst P-DNA with a left border-like sequence, a promoter, a desirednucleotide sequence, which is a sense-oriented nucleotide sequence fromthe leader sequence associated with the R1 gene, an antisense-orientedsequence from this leader sequence, a termination sequence, and a rightborder-like sequence. Upon expression, a leader-RNA duplex is producedthat reduces expression of the R1 gene, thereby reducing cold-inducedsweetening in the plant. In another embodiment, the desired sense- andantisense-oriented nucleotide sequences represent the trailer associatedwith the R1 gene. In a further embodiment, the sense- andantisense-oriented leader or trailer associated with R1 may be separatedby another polynucleotide sequence, referred to herein, as either anintron or a “spacer.”

In another preferred embodiment, the method comprises theLifeSupport-mediated transformation of a selected plant species with amodified P-DNA that is similar to the one described above but contains aleader- or trailer sequence associated with an alpha glucanphosphorylase gene.

In yet another preferred embodiment, the method comprises theLifeSupport-mediated transformation of a selected plant species with amodified P-DNA that contains an invertase inhibitor gene.

In another preferred embodiment, the modified P-DNA described in thepreceding paragraphs are used to reduce the accumulation of additionalundesirable products of the Maillard reaction, which occurs during theheating of carbohydrate-rich foods such as potato tubers. Theseundesirable products include advanced glycation end products (AGEs) thathave been associated with various pathologies.

The present invention also provides a method for increasing resistantstarch levels in the storage organs of plants and food crops.

In a preferred embodiment, the method comprises the LifeSupport-mediatedtransformation of a selected plant species with a modified P-DNA thatcontains an expression cassette for a fusion of the trailer sequencesassociated with the starch branching enzyme I and II genes.

The present invention also provides isolated nucleotide sequencescomprising the promoters of the potato GBSS gene and the potatoproteinase inhibitor gene, which are predominantly expressed in tubers.The isolated promoters have the nucleotide sequence shown in SEQ ID NO.:6 and SEQ ID NO.:40, respectively.

In one aspect, the present invention provides a method of modifying atrait of a selected plant comprising:

a. stably transforming cells from the selected plant with a desiredpolynucleotide, wherein the desired polynucleotide consists essentiallyof a nucleic acid sequence that is native to the selected plant, nativeto a plant from the same species, or is native to a plant that issexually interfertile with the selected plant,

b. obtaining a stably transformed plant from the transformed plant cellswherein the transformed plant contains the desired polynucleotide stablyintegrated into the genome and wherein the desired polynucleotidemodifies the trait.

In a preferred embodiment, the method further comprises co-transfectingthe plant cells with a selectable marker gene that is transientlyexpressed in the plant cells, and identifying transformed plant cells,and transformed plants obtained from the transformed plant cells,wherein the selectable marker gene is not stably integrated and thedesired polynucleotide is stably integrated into the genome.

In a preferred embodiment, the desired polynucleotide comprises a P-DNA,GBSS promoter, Ubi7 promoter, Ubi3 promoter, PIP promoter, modified PPOgene, invertase inhibitor gene, salt tolerance gene, R1-associatedleader, phosphorylase-associated leader, R1-associated trailer,SBE-associated trailers, Ubi-intron, GBSS spacer, UbiT.

In another preferred embodiment, a “plant” of the present invention is amonocotyledenous plant, selected from the group consisting of wheat,turf, turf grass, cereal, maize, rice, oat, wheat, barley, sorghum,orchid, iris, lily, onion, banana, sugarcane, sorghum, and palm.

In yet another embodiment, a “plant” of the present invention is adicotyledenous plant, selected from the group consisting of avacado,potato, tobacco, tomato, sugarbeet, broccoli, cassava, sweet potato,pepper, cotton, poinsetta, legumes, alfalfa, soybean, carrot,strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, andcactus.

In yet another embodiment, plants and plant cells of the presentinventive methods are transformed via Agrobacterium-mediatedtransformation. Preferably, the Agrobacterium-mediated transformationrelies on the use of at least one binary vector. In yet anotherembodiment, the Agrobacterium-mediated transformation method uses afirst binary vector and a second binary vector. In a preferredembodiment the first binary vector contains the desired polynucleotideand the second binary vector contains a selectable marker gene, whereinthe selectable marker gene is operably linked to a promoter and aterminator.

According to the present methods, the trait that is modified is selectedfrom the group consisting of enhanced health and nutritionalcharacteristics, improved storage, enhanced yield, enhanced salttolerance, enhanced heavy metal tolerance, increased drought tolerance,increased disease tolerance, increased insect tolerance, increasedwater-stress tolerance, enhanced cold and frost tolerance, enhancedcolor, enhanced sweetness, improved vigor, improved taste, improvedtexture, decreased phosphate content, increased germination, increasedmicronutrient uptake, improved starch composition, improved flowerlongevity.

The present invention also encompasses a plant made by the presentmethods.

In another aspect, the present invention provides a method of modifyinga trait in a selected plant comprising:

(a) identifying the trait to be modified;

(b) constructing a first polynucleotide consisting essentially of nativegenetic elements isolated from the selected plant, a plant from the samespecies, or a plant that is sexually interfertile with the selectedplant, wherein the native genetic elements are capable of modifying theexpression of a gene that controls the trait

(c) constructing a second polynucleotide comprising a selectable markergene that is operably linked to a promoter and a terminator;

(d) co-transfecting plant cells from the selected plant with the firstand second polynucleotides;

(e) selecting for the transient expression of the selectable markergene;

(f) screening for plant cells stably transformed with the firstpolynucleotide but do not contain the second DNA molecule integratedinto the genome; and

(g) obtaining a stably transformed plant from the transformed plantcells that exhibit a modified expression of the trait.

In one embodiment, the genetic elements comprise at least one of apromoter, sequence of interest, terminator, enhancer, intron, spacer, orregulatory elements. In another embodiment, method of claim 4, whereinthe plant cells are transfected with the first polynucleotide before thesecond polynucleotide or vice versa.

In one embodiment, the sequence of interest is a gene. In anotherembodiment, the gene is a mutated or wild-type polyphenol oxidase geneor a mutated or wild-type R1 gene. In one other embodiment, the sequenceof interest is a leader or trailer sequence, wherein the leader ortrailer sequence represents a sequence upstream or downstream of a genethat is native to the plant cell. In yet another embodiment, thesequence of interest comprises a sense-oriented leader sequence operablylinked to an antisense leader sequence. In another embodiment, thesequence of interest comprises a sense-oriented trailer sequenceoperably linked to an antisense trailer sequence. In another embodiment,the promoter is an inducible promoter. In another embodiment, theterminator is a yeast ADH terminator sequence.

According to the present invention, a leader construct comprises in 5′-to 3′-direction, a promoter, a sense-oriented leader sequence, theantisense sequence of the leader, and a terminator, wherein expressionof the leader construct produces a double-stranded RNA molecule thatfacilitates the down-regulation of expression of the gene to which it isassociated. In one other embodiment, the leader sequence is associatedwith, and located upstream of, the coding region of the PPO gene, the R1gene, an L-type phosphorylase gene, or an alpha glucan phosphorylasegene.

In another embodiment, the trailer construct comprises in 5′- to3′-direction, a promoter, a sense-oriented trailer sequence, theantisense sequence of the trailer, and a terminator, wherein expressionof the trailer construct produces a double-stranded RNA molecule thatfacilitates the down-regulation of expression of the gene to which it isassociated. In a preferred embodiment, the trailer sequence isassociated with, and located downstream of, the coding region of the PPOgene, the R1 gene, an L-type phosphorylase gene, or an alpha glucanphosphorylase gene.

The method further comprises exposing the plant cell to a second vectorthat comprises a marker element, wherein the marker is transientlyexpressed in the transformed plant and is not stably integrated into thegenome of the transformed plant. In one embodiment, the marker is aherbicide resistance gene, an antibiotic resistance gene, or NPTII.

Preferably, the plant cells are transformed via Agrobacterium-mediatedtransformation. In one embodiment, the Agrobacterium-mediatedtransformation relies on the use of at least one binary vector. In yetanother embodiment, the Agrobacterium-mediated transformation methoduses a first binary vector and a second binary vector. In one otherembodiment, the first binary vector carries the first polynucleotide andthe second binary vector carries the second polynucleotide.

The present invention provides another method of modifying theexpression of a gene in a selected plant comprising:

(a) identifying the functional gene;

(b) constructing a first polynucleotide consisting essentially of nativegenetic elements isolated from the selected plant, a plant of the samespecies as the selected plant, or a plant that is sexually interfertilewith the selected plant, wherein the native genetic elements are capableof modifying the expression of the gene;

(c) constructing a second polynucleotide comprising a functionalselectable marker gene;

(d) co-transfecting plant cells from the selected plant with the firstand second poylnucleotides;

(e) selecting for the transient expression of the selectable markergene;

(f) screening for plant cells stably transformed with the firstpolynucleotide but do not contain the second polynucleotide integratedinto the genome; and

(g) obtaining a transformed plant from the transformed plant cells thatexhibit modified expression of the gene.

Preferably, the plant cells are transformed via Agrobacterium-mediatedtransformation. In one embodiment, the Agrobacterium-mediatedtransformation relies on the use of at least one binary vector. In yetanother embodiment, the Agrobacterium-mediated transformation methoduses a first binary vector and a second binary vector. In one otherembodiment, the first binary vector carries the first polynucleotide andthe second binary vector carries the second polynucleotide.

In another embodiment, the first polynucleotide comprises at least oneof a P-DNA, GBSS promoter, Ubi7 promoter, Ubi3 promoter, PIP promoter,modified PPO gene, invertase inhibitor gene, salt tolerance gene,R1-associated leader, phosphorylase-associated leader, R1-associatedtrailer, SBE-associated trailers, Ubi-intron, GBSS spacer, UbiT.

In another embodiment, the second polynucleotide comprises at least oneof a selectable marker gene, an omega-mutated virD2 polynucleotide, acodA polynucleotide, and a codA::upp fusion polynucleotide.

The present invention also encompases a plant made by such method.

In one other embodiment, a transgenic plant is provided which exhibits amodified expression of a trait compared to the non-trasgenic plant fromwhich it was derived, wherein the transgenic plant is stably transformedwith a desired polynucleotide consisting essentially of native geneticelements isolated from the plant, a plant in the same species, or aplant that is sexually interfertile with the plant, and wherein thepolynucleotide modifies the expression of the trait.

In another preferred embodiment, the “plant” of the present invention isa monocotyledenous plant, selected from the group consisting of wheat,turf, turf grass, cereal, maize, rice, oat, wheat, barley, sorghum,orchid, iris, lily, onion, banana, sugarcane, sorghum, and palm.

In yet another embodiment, the “plant” of the present invention is adicotyledenous plant, selected from the group consisting of avacado,potato, tobacco, tomato, sugarbeet, broccoli, cassava, sweet potato,pepper, cotton, poinsetta, legumes, alfalfa, soybean, carrot,strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, andcactus.

In another embodiment, the trait is selected from the group consistingof enhanced health and nutritional characteristics, improved storage,enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance,increased drought tolerance, increased disease tolerance, increasedinsect tolerance, increased water-stress tolerance, enhanced cold andfrost tolerance, enhanced color, enhanced sweetness, improved vigor,improved taste, improved texture, decreased phosphate content, increasedgermination, increased micronutrient uptake, improved starchcomposition, improved flower longevity.

In another embodiment, the desired polynucleotide comprises at least oneof a P-DNA, GBSS promoter, Ubi7 promoter, Ubi3 promoter, PIP promoter,modified PPO gene, invertase inhibitor gene, salt tolerance gene,R1-associated leader, phosphorylase-associated leader, R1 -associatedtrailer, SBE-associated trailers, Ubi-intron, GBSS spacer, UbiT.

The present invention also encompasses an isolated, border-likenucleotide sequence ranging in size from 20 to 100 bp that sharesbetween 52% and 96% sequence identity with a T-DNA border sequence fromAgrobacterium tumafaciens. In a preferred embodiment, the isolatednucleotide sequence is isolated from a monocotyledenous plant, selectedfrom the group consisting of wheat, turf, turf grass, cereal, maize,rice, oat, wheat, barley, sorghum, orchid, iris, lily, onion, banana,sugarcane, sorghum, and palm. In another embodiment, the nucleotidesequence is isolated from a dicotyledenous plant selected from the groupconsisting of potato, tobacco, tomato, sugarbeet, broccoli, cassava,sweet potato, pepper, cotton, poinsetta, legumes, alfalfa, soybean,carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash,daisy, and cactus.

In yet another embodiment, the isolated nucleotide sequence is isolatedfrom potato, and has a nucleotide sequence shown in either SEQ ID NO. 94or 95. In a preferred embodiment, the isolated nucleotide sequenceshares 52% sequence identity with a T-DNA border sequence fromAgrobacterium tumafaciens. The present invention encompasses a vectorthat comprises such nucleotide sequences.

The present invention also provides method of making a plant stablytransformed with a desired polynucleotide comprising:

(a) isolating a P-DNA that is flanked by border-like sequences from theplant wherein the border-like sequences share between 52% and 96%sequence identity with an Agrobacterium tumafaciens T-DNA bordersequence;

(b) inserting the desired polynucleotide between the P-DNA border-likesequences to form a P-DNA construct; and

(c) transforming aa plant cell from the plant with the P-DNA construct;and

(d) recovering a plant from the transformed plant cell stablytransformed with the P-DNA construct.

In one embodiment, the P-DNA construct is carried on a vector comprisedof a backbone integration marker gene and transformed plant cells areselected that do not contain the backone integration marker gene. Inanother embodiment, the backbone integration marker gene is a cytokiningene. In another embodiment, plant shoots are not selected that exhibita cytokinin-overproducing phenotype. In yet another embodiment, thebacknone integraton marker gene is the IPT gene, and plant shoots arenot selected that exhibit an abnormal phenotype or cannot develop roots.

In one other embodiment, the plant cells are from a monocotyledenousplant selected from the group consisting of wheat, turf, turf grass,cereal, maize, rice, oat, wheat, barley, sorghum, orchid, iris, lily,onion, banana, sugarcane, sorghum, and palm.

In another embodiment, the plant cells are from a dicotyledenous plantselected from the group consisting of potato, tobacco, tomato,sugarbeet, broccoli, cassava, sweet potato, pepper, cotton, poinsetta,legumes, alfalfa, soybean, carrot, strawberry, lettuce, oak, maple,walnut, rose, mint, squash, daisy, and cactus.

Preferably, the plant cells are transformed via Agrobacterium-mediatedtransformation. In one embodiment, the Agrobacterium-mediatedtransformation relies on the use of at least one binary vector. In yetanother embodiment, the Agrobacterium-mediated transformation methoduses a first binary vector and a second binary vector. In one otherembodiment, the first binary vector carries the first polynucleotide andthe second binary vector carries the second polynucleotide. In onefurther embodiment, the second binary vector comprises at least one of anegative selectable marker gene and an omega-mutated virD2 gene, whereinthe negative selectable marker gene is positioned within the right T-DNAborder and the left T-DNA border, and wherein the omega-mutated virD2gene is positioned within the backbone of the second binary vector. In apreferred embodiment, the second binary vector comprises both a negativeselectable marker gene positioned within the right T-DNA border and theleft T-DNA border, and an omega-mutated virD2 gene positioned within thebackbone of the second binary vector.

The present invention also provides a P-DNA consisting essentially of,in the 5′- to 3′-direction, a first T-DNA border-like sequence, apromoter, a desired polynucleotide sequence operably linked to thepromoter, a terminator, and a second T-DNA border-like sequence, whereinthe border-like sequences have less than 100% sequence identity withT-DNA border sequences

In a preferred embodiment, the T-DNA border-like sequences, thepromoter, the desired polynucleotide, and the terminator, are allisolated from the same plant, the same plant species, or plants that aresexually interfertile.

In another embodiment, the P-DNA further consists essentially of aselectable marker gene.

In yet another embodiment, the T-DNA border-like sequences, thepromoter, the desired polynucleotide, the terminator and the selectablemarker gene, are all isolated from the same plant, the same plantspecies, or plants that are sexually interfertile.

In yet another embodiment, the desired polynucleotide sequence in theP-DNA is a sequence upstream or downstream of the coding region of agene, wherein the upstream sequence is a leader sequence, and whereinthe downstream sequence is a trailer sequence. In this embodiment, theT-DNA border-like sequences, the promoter, the leader sequence, thetrailer sequence, the terminator and the selectable marker gene are allisolated from the same plant, the same plant species, or plants that aresexually interfertile.

In another embodiment, vectors comprising such P-DNA constructs areprovided by the present invention.

In another embodiment, the promoter is a regulatable promoter. In yetanother embodiment, the regulatable promoter is sensitive totemperature. In a preferred embodiment, the regulatable promoter is awheat wcs120 promoter. In another embodiment, the promoter is undertemporal regulation. In yet another embodiment, the promoter is acarboxylase promoter. In a further embodiment, the carboxylase promoteris a maize carboxylase promoter.

The promoter maybe regulated by any one of abscisic acid, wounding,methyl jasmonate or gibberellic acid. In another embodiment, thepromoter is a promoter selected from either a Rab 16A gene promoter, anα-amylase gene promoter or a pin2 gene promoter. In yet anotherembodiment, the promoter is a tissue-specific promoter.

In one other embodiment, the leader sequence is a part of a5′-untranslated region of a gene that is endogenous to a cell of theselected plant species. In another embodiment, the 5′-untranslatedregion is upstream of a start codon of a gene that is selected from thegroup consisting of a PPO gene, an R1 gene, a HOS1 gene, aS-adenosylhomocysteine hydrolase gene, a class II cinnamate4-hydroxylase gene, a cinnamoyl-coenzyme A reductase gene, a cinnamoylalcohol dehydrogenase gene, a caffeoyl coenzyme A O-methyltransferasegene, an actin depolymerizing factor gene, a Nin88 gene, a Lol p 5 gene,an allergen gene, a P450 hydroxylase gene, an ADP-glucosepyrophosphorylase gene, a proline dehydrogenase gene, anendo-1,4-beta-glucanase gene, a zeaxanthin epoxidase gene, and a1-aminocyclopropane-1-carboxylate synthase gene.

In another embodiment, the trailer sequence is a part of the3′-untranslated region of a gene that is downstream of a terminationcodon of a gene selected from the group consisting of a PPO gene, an R1gene, a HOS1 gene, a S-adenosylhomocysteine hydrolase gene, a class IIcinnamate 4-hydroxylase gene, a cinnamoyl-coenzyme A reductase gene, acinnamoyl alcohol dehydrogenase gene, a caffeoyl coenzyme AO-methyltransferase gene, an actin depolymerizing factor gene, a Nin88gene, a Lol p 5 gene, an allergen gene, a P450 hydroxylase gene, anADP-glucose pyrophosphorylase gene, a proline dehydrogenase gene, anendo-1,4-beta-glucanase gene, a zeaxanthin epoxidase gene, and a1-aminocyclopropane-1-carboxylate synthase gene.

The present vector may further comprise a spacer element that is eitheran Ubi intron sequence or a GBSS spacer sequence. In another embodiment,the vector comprises a terminator that is a Ubi3 terminator sequence ora 3′-untranslated region of an endogenous plant gene.

In another embodiment, the vector comprises a selectable marker geneoperably linked to a constitutive promoter and a Cre gene operablylinked to an inducible promoter, wherein the selectable marker gene andthe Cre gene are flanked by a first recombinase recognition site and asecond recombinase recognition site. In another embodiment, the firstrecombinase recognition site and the second recombinase recognition siteare lox sites.

In another embodiment, the inducible promoter is a temperature-sensitivepromoter, a chemically-induced promoter, or a temporal promoter. In yetanother embodiment, the inducible promoter is a Ha hsp17.7 G4 promoter,a wheat wcs120 promoter, a Rab 16A gene promoter, an α-amylase genepromoter, a pin2 gene promoter, a carboxylase promoter. In yet anotherpreferred embodiment, further comprises a plant-derived marker gene. Inanother preferred embodiment, the plant-derived marker gene is anenolpyruvul-3-phosphoshikimic acid synthase gene.

In another aspect of the present invention, a method for modifying aplant cell is provided, comprising integrating a P-DNA sequence into thegenome of a plant cell, wherein the P-DNA consists essentially of, inthe 5′- to 3′-direction, a first T-DNA border-like sequence, a promoter,a desired polynucleotide sequence operably linked to the promoter, aterminator, and a second T-DNA border-like sequence, wherein theborder-like sequences have less than 100% sequence identity with T-DNAborder sequences, and wherein the T-DNA border-like sequences, thepromoter, the desired polynucleotide, and terminator, are all isolatedfrom or native to the genome of the plant cell, wherein the desiredpolynucleotide comprises sense and antisense seqeunces of a leadersequence or trailer sequence that are associated with the upstream ordownstream non-coding regions of a gene in the plant, and whereinexpression of the desired polynucleotide produces a double-stranded RNAtranscript that targets the gene associated with the desiredpolynucleotide, thereby modifying the plant cell.

The present invention also encompasses a method for modifying a plant,comprising:

(i) transfecting at least one cell in the plant with the vector of thepresent invention;

(ii) selecting a cell expressing the functional selectable marker;

(iii) isolating the cell expressing the functional selectable marker;

(iii) inducing the expression of the functional Cre gene in the isolatedcell;

(iv) culturing the isolated cell; and

(ii) observing the phenotype of cultured cells;

wherein a phenotype that is different to an untransfected plant cellindicates that the target plant cell has been modified.

In a preferred embodiment the selecting step of this and other methodsof the present invention is performed by identifying which cells areresistant to an antibiotic.

In another aspect, a method for identifying a target plant cell whosegenome contains a P-DNA, comprises co-transfecting a plant target cellwith the vector of the present invention and a secondAgrobacterium-derived vector that comprises a marker gene flanked by aT-DNA left border and a T-DNA right border and a omega-mutated virD2gene, wherein the P-DNA is integrated into the genome of the planttarget cell, and wherein no part of the second Agrobacterium-derivedvector is integrated into the genome of the plant target cell. In apreferred embodiment, the marker in the second Agrobacterium-derivedvector is a neomycin phosphotransferase gene.

In another aspect, the method for identifying a target plant cell whosegenome contains at least a part of an integration cassette is provided,further comprises selecting cells that survive temporary growth on akanamycin-containing media, wherein the genomes of the selected cellscontain only the integration cassette. In one embodiment, the targetplant cell is within a plant. A plant comprising at least one cell whosegenome comprises such a P-DNA is also encompassed by the presentinvention.

The present invention also encompasses a plant comprising at least onecell whose genome is artificially manipulated to contain onlyplant-derived nucleic acids, wherein no cells of the plant containforeign nucleic acids integrated into the cell genome.

The present invention also encompasses a polynucleotide comprising thepolynucleotide sequence of SEQ ID NO. 93, wherein the polynucleotide isbetween 20 and 80 nucleotides in length. In one embodiment, thepolynucleotide is between 21 and 70 nucleotides in length, between 22and 50 nucleotides in length, between 23 and 40 nucleotides in length,or between 24 and 30 nucleotides in length.

In another aspect, the invention encompasses a tuber-specific promoteras shown in SEQ ID NO. 40.

The present invention also encompasses an Agrobacterium-based method ofmaking transgenic plant cells that do not contain a selectable markergene stably integrated in nuclear DNA comprising:

-   -   a. constructing a first binary vector comprised of a        polynucleotide consisting essentially of a desired functional        gene operably linked to T-DNA borders or T-DNA border-like        sequences at the 5′ and 3′ ends of the desired functional gene;    -   b. constructing a second binary vector comprised of a functional        selectable marker gene operably linked to T-DNA borders or T-DNA        border-like sequences at the 5′ and 3′ ends of the functional        selectable marker gene;    -   c. incubating plants cells with:        -   i. an Agrobacterium strain carrying the first and the second            binary vectors; or        -   ii. a first Agrobacterium strain carrying the first binary            vector and a second Agrobacterium strain carrying the second            binary vector;    -   d. selecting plant cells wherein the desired functional gene is        integrated into plant nuclear DNA without integration of the        selectable marker gene into plant nuclear DNA following        incubation for an appropriate time period on a medium containing        an appropriate selection agent.

In a preferred embodiment, the selectable marker gene is a herbicideresistance gene or an antibiotic resistance gene. In another preferredembodiment, the antibiotic resistance gene is the nNPTII gene. Inanother embodiment, the antibiotic resistance gene is the npt IIstructural gene operably linked to the promoter from the Ubiquitin-7gene and the termninator from yeast alcohol dehydrogenase 1 (ADH1) gene.According to this method, the plant cells are first incubated with thefirst Agrobacterium strain and then subsequently incubated with thesecond Agrobacterium strain or vice versa.

In a preferred embodiment, the first binary vector further comprises abinary integration marker gene that can be used to detect plant cellsstably transformed with binary vector backbone sequences. In anotherembodiment, the binary vector integration marker gene is selected fromthe group consisting of herbicide resistance gene, antibiotic resistancegene, or NPTII. In yet another embodiment, the second binary vectorfurther comprises a gene fusion between the bacterial cytosine deaminase(codA) and uracil phophoribsyltransferase (upp) genes, which is insertedbetween the T-DNA or T-DNA border-like sequences, and plant cells areexposed to 5-fluorocytosine following incubation with the first andsecond Agrobacterium strains in order to select against those plantcells transformed with the second binary vector.

In yet another embodiment, the secondary binary vector further comprisesa gene that reduces the probability of backbone integration. In oneembodiment, such a gene is the omega-mutated virD2 gene, wherein theomega-mutated virD2 gene reduces the frequency of integration of theselectable marker gene into the plant nuclear DNA.

The present invention also encompasses an isolated nucleotide sequencecomprising the GBSS promoter isolated from S. tuberosum. In a preferredembodiment, this isolated nucleotide sequence has the nucleotidesequence that is SEQ ID. NO. 6 or 13.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic illustrations of some P-DNA vectors used in thepresent invention. P-DNA region is indicated as grey box.“ipt”=expression cassette for the ipt gene; “npt”=expression cassettefor the nptII gene; “mPPO”=expression cassette for a modified PPO gene;“INH”=expression cassette for an invertase inhibitor gene;“GUS”=expression cassette for the GUS gene; “LPPO”=expression cassettefor a sense and antisense copy of the leader associated with a PPO gene;“LPH”=expression cassette for a sense and antisense copy of the leaderassociated with a phosphorylase gene; “Alf”=expression cassette for apotato Alfin homolog. See text for details.

FIG. 2. Alignment of potato and tobacco invertase inhibitor proteins.“St”=Solanum tuberosum (potato); “Nt”=Nicotiana tabacum (tobacco)

FIG. 3. Gene-free expression cassettes

FIG. 4. Alignment of trailers associated with various PPO genes.

FIG. 5. Schematic illustrations of some LifeSupport vectors used in thepresent invention. “codA” is an expression cassette for the codA gene;“codA::upp” is an expression cassette for the codA gene fused to upp;“ΩvirD2” is an expression cassette for the ΩvirD2 gene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The “precise breeding” strategy of the present invention improves theagronomic performance, nutritional value, and health characteristics ofplants and crops without introducing unknown nucleic acid, or nucleicacid from a foreign species into a plant species genome, and withoutproducing undesirable phenotypes or harmful side-effects.

Thus, the present invention provides a transgenic plant, and methods formaking such a plant that do not integrate nucleic acid from non-plantspecies into that plant's genome. Nucleic acids, promoters, regulatoryelements, other non-coding gene sequences, markers, polynucleotides, andgenes that are integrated into the selected plant genome are allpreferably isolated from the plant that is to be transformed, plants ofthe same species to be transformed, or plants that are sexuallyinterfertile with the plant to be transformed. Such “native” nucleicacids can be mutated, modified or cojoined with other native nucleicacids in an expression cassette and reintegrated into the selected plantgenome, according to the methods described herein. Accordingly, thegenotype and phenotype of the transgenic plant is altered using onlythat selected plant's own nucleic acid, or using nucleic acid from aplant that is sexually compatible with the selected plant.

To facilitate the production of such transgenic plants, the presentinvention makes use of the fact that not all T-DNA vectors used inAgrobacterium-mediated transformation are actually integrated into theplant genome; i.e., while a vector may be taken up by the plant cell, anactual integration event may not occur. According to the presentinvention, one may use such a vector to carry a selectable marker geneinto a plant cell. Plant cells can then be screened to determine whetherthe marker has been stably integrated into the plant genome bydetermining for how long the marker gene is expressed. Accordingly,plant cells that only transiently express the selectable marker gene aredesired because they represent cells that took up, but did not integrateinto their genomes, the selectable marker gene.

Thus, by co-transforming a plant with such a “marker vector” and alsowith another vector that contains the desired native gene orpolynucleotide, one can select plant cells that took up both vectorsand, from those, determine which cells possess genomes that contain onlythe desired gene or polynucleotide. The “marker vector” can be modifiedto further reduce the possibility that the marker will be integratedinto the plant genome. The present invention provides such “markervectors” in the form of “LifeSupport” vectors.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein, and the laboratory procedures in cell culture, moleculargenetics, and nucleic acid chemistry and hybridization described herein,are those well known and commonly employed in the art. Standardtechniques are used for recombinant nucleic acid methods, polynucleotidesynthesis, microbial culture, cell culture, tissue culture,transformation, transfection, transduction, analytical chemistry,organic synthetic chemistry, chemical syntheses, chemical analysis, andpharmaceutical formulation and delivery. Generally, enzymatic reactionsand purification and/or isolation steps are performed according to themanufacturers' specifications. The techniques and procedures aregenerally performed according to conventional methodology disclosed, forexample, in Molecular cloning a laboratory manual, 2d ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and Currentprotocols in molecular biology, John Wiley & Sons, Baltimore, Md.(1989).

Amino acid sequence: as used herein, includes an oligopeptide, peptide,polypeptide, or protein and fragments thereof, that are isolated from,native to, or naturally occurring in a plant, or are synthetically madebut comprise the nucleic acid sequence of the endogenous counterpart.

Artificially manipulated: as used herein, “artificially manipulated”means to move, arrange, operate or control by the hands or by mechanicalmeans or recombinant means, such as by genetic engineering techniques, aplant or plant cell, so as to produce a plant or plant cell that has adifferent biological, biochemical, morphological, or physiologicalphenotype and/or genotype in comparison to unmanipulated,naturally-occurring counterpart.

Asexual propagation: producing progeny by generating an entire plantfrom leaf cuttings, stem cuttings, root cuttings, tuber eyes, stolons,single plant cells protoplasts, callus and the like, that does notinvolve fusion of gametes.

Backbone: nucleic acid sequence of a binary vector that excludes theT-DNA or P-DNA sequence intended for transfer.

Border and Border-like sequences: “border sequences” are specificAgrobacterium-derived sequences. Typically, a left border sequence and aright border sequence flank a T-DNA and they both function asrecognition sites for virD2-catalyzed nicking reactions. Such activityreleases nucleic acid that is positioned between such borders. See Table2 below for examples of border sequences. The released nucleic acid,complexed with virD2 and virE2, is targeted to plant cell nuclei wherethe nucleic acid is often integrated into the genome of the plant cell.Usually, two border sequences, a left-border and a right-border, areused to integrate a nucleotide sequence that is located between theminto another nucleotide sequence. It is also possible to use only oneborder, or more than two borders, to accomplish integration of a desirednucleic acid in such fashion.

According to the present invention, a “border-like” sequence is isolatedfrom the selected plant species that is to be modified, or from a plantthat is sexually-compatible with the plant species to be modified, andfunctions like the border sequences of Agrobacterium. That is, aborder-like sequence of the present invention promotes and facilitatesthe integration of a polynucleotide to which it is linked. A plant-DNA,i.e., P-DNA, of the present invention preferably contains border-likesequences.

A border-like sequence of a P-DNA is between 5-100 bp in length, 10-80bp in length, 15-75 bp in length, 15-60 bp in length, 15-50 bp inlength, 15-40 bp in length, 15-30 bp in length, 16-30 bp in length,20-30 bp in length, 21-30 bp in length, 22-30 bp in length, 23-30 bp inlength, 24-30 bp in length, 25-30 bp in length, or 26-30 bp in length.

The border-like sequences of the present invention can be isolated fromany plant, such as from potato and wheat. See SEQ ID NO. 1 and SEQ IDNO. 34, for sequences which contain, at either end, the border-likesequences isolated from potato and wheat respectively. Thus, a P-DNAleft and right border sequences of use for the present invention areisolated from and/or native to the genome of a plant that is to bemodified. A P-DNA border-like sequence is not identical in nucleotidesequence to any known Agrobacterium-derived T-DNA border sequence. Thus,a P-DNA border-like sequence may possess 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides that aredifferent from a T-DNA border sequence from an Agrobacterium species,such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. That is,a P-DNA border, or a border-like sequence of the present invention hasat least 95%, at least 90%, at least 80%, at least 75%, at least 70%, atleast 60% or at least 50% sequence identity with a T-DNA border sequencefrom an Agrobacterium species, such as Agrobacterium tumefaciens orAgrobacterium rhizogenes, but not 100% sequence identity. As usedherein, the descriptive terms “P-DNA border” and “P-DNA border-like” areexchangeable.

A native P-DNA border sequence is greater than or equal to 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%,83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%,69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%,55%, 54%, 53%, 52%, 51% or 50% similar in nucleotide sequence to aAgrobacterium a T-DNA border sequence. A border-like sequence can,therefore, be isolated from a plant genome and be modified or mutated tochange the efficiency by which they are capable of integrating anucleotide sequence into another nucleotide sequence. Otherpolynucleotide sequences may be added to or incorporated within aborder-like sequence of the present invention. Thus, a P-DNA left borderor a P-DNA right border may be modified so as to possess 5′- and3′-multiple cloning sites, or additional restriction sites. A P-DNAborder sequence may be modified to increase the likelihood that backboneDNA from the accompanying vector is not integrated into the plantgenome.

Table 2 below depicts the sequences of known T-DNA border sequences andsequences identified herein as border-like sequences. None of thesequences identified as “border-like” in Table 2 have been identifiedpreviously as having a T-DNA border-like structure. The potatoborder-like sequences were isolated by the present inventive methodsusing degenerate primers in polymerase chain reactions from potatogenomic DNA. The present invention encompasses the use of any P-DNAborder-like sequence for transferring a cojoined polynucleotide into thegenome of a plant cell.

Indeed, the present invention encompasses any border-like sequence thathas the nucleic acid sequence structure of SEQ ID NO. 93: ANGATNTATN6GT(SEQ ID NO. 93), where “N” is any nucleotide, such as those representedby “A,” “G,” “C,” or “T.” This sequence represents the consensussequence of border-like nucleic acids identified by the presentinvention. TABLE 2 “Border” and “Border-Like” sequences AgrobacteriumT-DNA borders TGACAGGATATATTGGCGGGTAAAC (SEQ ID NO.41) Agrobacteriumnopaline strains (RB) TGGCAGGATATATTGTGGTGTAAAC (SEQ ID NO.42)Agrobacterium nopaline strains (LB) TGGCAGGATATATACCGTTGTAATT (SEQ IDNO.43) Agrobacterium octopine strains (RB) CGGCAGGATATATTCAATTGTAATT(SEQ ID NO.44) Agrobacterium octopine strains (LB)TGGTAGGATATATACCGTTGTAATT (SEQ ID NO.45) LB mutantTGGCAGGATATATGGTACTGTAATT (SEQ ID NO.46) LB mutantYGRYAGGATATATWSNVBKGTAAWY (SEQ ID NO.47) Border motifCGGCAGGATATATCCTGATGTAAAT (SEQ ID NO.48) R. leguminosarumTGGCAGGAGTTATTCGAGGGTAAAC (SEQ ID NO.49) T. tengcongensisTGACAGGATATATCGTGATGTCAAC (SEQ ID NO.50) Arabidopsis thalianaGGGAAGTACATATTGGCGGGTAAAC (SEQ ID NO.51) A. thaliana CHR1v07142002TTACAGGATATATTAATATGTATGA (SEQ ID NO.52) Oryza sativa AC078894TAACATGATATATTCCCTTGTAAAT (SEQ ID NO.53) Homo sapiens clone HQ0089TGACAGGATATATGGTAATGTAAAC (SEQ ID NO.54) potato (left border sequence)*TGGCAGGATATATACCGATGTAAAC (SEQ ID NO.55) potato (right border sequence)*Y = C or T; R = A or G; K = G or T; M = A or C; W = A or T; S = C or G;V = A, C, or G; B = C, G, or T.The accession numbers for the border-like sequences are: Oryza sativachromosome 10 BAC OSJNBa0096G08 genomic sequence (AC078894.11);Arabidopsis thaliana chromosome 3 (NM_114337.1); Arabidopsis thalianachromosome 1 (NM_105664.1); T. tengcongensis strain MB4T, section 118 of244 of the complete genome (AE013091.1); Homo sapiens clone HQ0089(AF090888.1); Rhizobium Clone: rhiz98e12.qlk.*potato left and right border sequences were obtained and isolatedaccording to the presently-described inventive methods.

Carrier DNA: a “carrier DNA” is a DNA segment that is used to carrycertain genetic elements and deliver them into a plant cell. Inconventional foreign DNA transfer, this carrier DNA is often the T-DNAof Agrobacterium, delineated by border sequences. The carrier DNAdescribed here is obtained from the selected plant species to bemodified and contains ends that may be structurally and functionallydifferent from T-DNA borders but shares with such T-DNAs the ability tosupport both DNA transfer from Agrobacterium to the nuclei of plantcells or certain other eukaryotes and the subsequent integration of thisDNA into the genomes of such eukaryotes.

Consisting essentially of: a composition “consisting essentially of”certain elements is limited to the inclusion of those elements, as wellas to those elements that do not materially affect the basic and novelcharacteristics of the inventive composition. Thus, so long as thecomposition does not affect the basic and novel characteristics of theinstant invention, that is, does not contain foreign DNA that is notfrom the selected plant species or a plant that is sexually compatiblewith the selected plant species, then that composition may be considereda component of an inventive composition that is characterized by“consisting essentially of” language.

Degenerate primer: a “degenerate primer” is an oligonucleotide thatcontains sufficient nucleotide variations that it can accommodate basemismatches when hybridized to sequences of similar, but not exact,homology.

Dicotyledon (dicot): a flowering plant whose embryos have two seedleaves or cotyledons. Examples of dicots include, but are not limitedto, tobacco, tomato, potato, sweet potato, cassava, legumes includingalfalfa and soybean, carrot, strawberry, lettuce, oak, maple, walnut,rose, mint, squash, daisy, and cactus.

Regulatory sequences: refers to those sequences which are standard andknown to those in the art, that may be included in the expressionvectors to increase and/or maximize transcription of a gene of interestor translation of the resulting RNA in a plant system. These include,but are not limited to, promoters, peptide export signal sequences,introns, polyadenylation, and transcription termination sites. Methodsof modifying nucleic acid constructs to increase expression levels inplants are also generally known in the art (see, e.g. Rogers et al., 260J. Biol. Chem. 3731-38, 1985; Cornejo et al., 23 Plant Mol. Biol. 567:81, 1993). In engineering a plant system to affect the rate oftranscription of a protein, various factors known in the art, includingregulatory sequences such as positively or negatively acting sequences,enhancers and silencers, as well as chromatin structure may have animpact. The present invention provides that at least one of thesefactors may be utilized in engineering plants to express a protein ofinterest. The regulatory sequences of the present invention are nativegenetic elements, i.e., are isolated from the selected plant species tobe modified.

Foreign: “foreign,” with respect to a nucleic acid, means that thatnucleic acid is derived from non-plant organisms, or derived from aplant that is not the same species as the plant to be transformed or isnot derived from a plant that is not interfertile with the plant to betransformed, does not belong to the species of the target plant.According to the present invention, foreign DNA or RNA representsnucleic acids that are naturally occurring in the genetic makeup offungi, bacteria, viruses, mammals, fish or birds, but are not naturallyoccurring in the plant that is to be transformed. Thus, a foreignnucleic acid is one that encodes, for instance, a polypeptide that isnot naturally produced by the transformed plant. A foreign nucleic aciddoes not have to encode a protein product. According to the presentinvention, a desired transgenic plant is one that does not contain anyforeign nucleic acids integrated into its genome.

Native genetic elements, on the other hand, can be incorporated andintegrated into a selected plant species genome according to the presentinvention. Native genetic elements are isolated from plants that belongto the selected plant species or from plants that are sexuallycompatible with the selected plant species. For instance, native DNAincorporated into cultivated potato (Solanum tuberosum) can be derivedfrom any genotype of S. tuberosum or any genotype of a wild potatospecies that is sexually compatible with S. tuberosum (e.g., S.demissum).

Gene: “gene” refers to the coding region and does not include nucleotidesequences that are 5′- or 3′- to that region. A functional gene is thecoding region operably linked to a promoter or terminator.

Genetic rearrangement: refers to the reassociation of genetic elementsthat can occur spontaneously in vivo as well as in vitro which introducea new organization of genetic material. For instance, the splicingtogether of polynucleotides at different chromosomal loci, can occurspontaneously in vivo during both plant development and sexualrecombination. Accordingly, recombination of genetic elements bynon-natural genetic modification techniques in vitro is akin torecombination events that also can occur through sexual recombination invivo.

In frame: nucleotide triplets (codons) are translated into a nascentamino acid sequence of the desired recombinant protein in a plant cell.Specifically, the present invention contemplates a first nucleic acidlinked in reading frame to a second nucleic acid, wherein the firstnucleotide sequence is a gene and the second nucleotide is a promoter orsimilar regulatory element.

Integrate: refers to the insertion of a nucleic acid sequence from aselected plant species, or from a plant that is from the same species asthe selected plant, or from a plant that is sexually compatible with theselected plant species, into the genome of a cell of a selected plantspecies. “Integration” refers to the incorporation of only nativegenetic elements into a plant cell genome. In order to integrate anative genetic element, such as by homologous recombination, the presentinvention may “use” non-native DNA as a step in such a process. Thus,the present invention distinguishes between the “use of” a particularDNA molecule and the “integration” of a particular DNA molecule into aplant cell genome.

Introduction: as used herein, refers to the insertion of a nucleic acidsequence into a cell, by methods including infection, transfection,transformation or transduction.

Isolated: “isolated” refers to any nucleic acid or compound that isphysically separated from its normal, native environment. The isolatedmaterial may be maintained in a suitable solution containing, forinstance, a solvent, a buffer, an ion, or other component, and may be inpurified, or unpurified, form.

Leader: Transcribed but not translated sequence preceding (or 5′ to) agene.

LifeSupport Vector: a LifeSupport vector is a construct that contains anexpressable selectable marker gene, such as a neomycinphosphotransferase marker, that is positioned between T-DNA orT-DNA-like borders. The LifeSupport vector may be modified to limitintegration of such a marker, as well as other polynucleotides, that aresituated between the border or border-like sequences, into a plantgenome. For instance, a LifeSupport vector may comprise a mutated virD2,codA::upp fusion, or any combination of such genetic elements. Thus, amodified virD2 protein will still support T-DNA transfer to plant nucleibut will limit the efficiency of a subsequent genomic integration ofT-DNAs (Shurvinton et al., Proc Natl Acad Sci USA, 89: 11837-11841,1992; Mysore et al., Mol Plant Microbe Interact, 11: 668-683, 1998).Alternatively, codA::upp gene fusion can be used as negative selectablemarker prior to regeneration. In one preferred construct, theLifeSupport vector comprises the npt marker operably linked to the yeastADH terminator element.

Monocotyledon (monocot): a flowering plant whose embryos have onecotyledon or seed leaf. Examples of monocots include, but are notlimited to turf grass, maize, rice, oat, wheat, barley, sorghum, orchid,iris, lily, onion, and palm.

Native: a “native” genetic element refers to a nucleic acid thatnaturally exists in, orginates from, or belongs to the genome of a plantthat is to be transformed. Thus, any nucleic acid, gene, polynucleotide,DNA, RNA, mRNA, or cDNA molecule that is isolated either from the genomeof a plant or plant species that is to be transformed or is isolatedfrom a plant or species that is sexually compatible or interfertile withthe plant species that is to be transformed, is “native” to, i.e.,indigenous to, the plant species. In other words, a native geneticelement represents all genetic material that is accessible to plantbreeders for the improvement of plants through classical plant breeding.Any variants of a native nucleic acid also are considered “native” inaccordance with the present invention. In this respect, a “native”nucleic acid may also be isolated from a plant or sexually compatiblespecies thereof and modified or mutated so that the resultant variant isgreater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%,76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,62%, 61%, or 60% similar in nucleotide sequence to the unmodified,native nucleic acid isolated from a plant. A native nucleic acid variantmay also be less than about 60%, less than about 55%, or less than about50% similar in nucleotide sequence.

A “native” nucleic acid isolated from a plant may also encode a variantof the naturally occurring protein product transcribed and translatedfrom that nucleic acid. Thus, a native nucleic acid may encode a proteinthat is greater than or equal to 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%,77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%,63%, 62%, 61%, or 60% similar in amino acid sequence to the unmodified,native protein expressed in the plant from which the nucleic acid wasisolated.

Naturally occurring nucleic acid: this phrase means that the nucleicacid is found within the genome of a selected plant species and may be aDNA molecule or an RNA molecule. The sequence of a restriction site thatis normally present in the genome of a plant species can be engineeredinto an exogenous DNA molecule, such as a vector or oligonucleotide,even though that restriction site was not physically isolated from thatgenome. Thus, the present invention permits the synthetic creation of anucleotide sequence, such as a restriction enzyme recognition sequence,so long as that sequence is naturally occurring in the genome of theselected plant species or in a plant that is sexually compatible withthe selected plant species that is to be transformed.

Operably linked: combining two or more molecules in such a fashion thatin combination they function properly in a plant cell. For instance, apromoter is operably linked to a structural gene when the promotercontrols transcription of the structural gene.

P-DNA: according to the present invention, P-DNA (“plant-DNA”) isisolated from a plant genome and comprises at each end, or at only oneend, a T-DNA border-like sequence. The border-like sequence preferablyshares at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 90% or at least 95%, but less than 100% sequence identity,with a T-DNA border sequence from an Agrobacterium species, such asAgrobacterium tumefaciens or Agrobacterium rhizogenes. Thus, P-DNAs canbe used instead of T-DNAs to transfer a nucleotide sequence fromAgrobacterium to another polynucleotide sequence. The P-DNA may bemodified to facilitate cloning and should preferably not naturallyencode proteins or parts of proteins. The P-DNA is characterized in thatit contains, at each end, at least one border sequence, referred to aseither a “P-DNA border sequence” or “P-DNA border-like sequence,” whichare interexchangeable terms. See the definition of a “border sequence”and “border-like” above. A P-DNA may also be regarded as a “T-DNA-like”sequence, see definition below.

Plant: includes angiosperms and gymnosperms such as potato, tomato,tobacco, alfalfa, lettuce, carrot, strawberry, sugarbeet, cassava, sweetpotato, soybean, maize, turf grass, wheat, rice, barley, sorghum, oat,oak, eucalyptus, walnut, and palm. Thus, a plant may be a monocot or adicot. The word “plant,” as used herein, also encompasses plant cells,seed, plant progeny, propagule whether generated sexually or asexually,and descendents of any of these, such as cuttings or seed. Plant cellsinclude suspension cultures, callus, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,seeds and microspores. Plants may be at various stages of maturity andmay be grown in liquid or solid culture, or in soil or suitable media inpots, greenhouses or fields. Expression of an introduced leader, traileror gene sequences in plants may be transient or permanent. A “selectedplant species” may be, but is not limited to, a species of any one ofthese “plants.”

Precise breeding: refers to the improvement of plants by stableintroduction of nucleic acids, such as native genes and regulatoryelements isolated from the selected plant species, or from another plantin the same species as the selected plant, or from species that aresexually compatible with the selected plant species, into individualplant cells, and subsequent regeneration of these genetically modifiedplant cells into whole plants. Since no unknown or foreign nucleic acidis permanently incorporated into the plant genome, the inventivetechnology makes use of the same genetic material that is alsoaccessible through conventional plant breeding.

Plant species: the group of plants belonging to various officially namedplant species that display at least some sexual compatibility.

Plant transformation and cell culture: broadly refers to the process bywhich plant cells are genetically modified and transferred to anappropriate plant culture medium for maintenance, further growth, and/orfurther development.

Recombinant: as used herein, broadly describes various technologieswhereby genes can be cloned, DNA can be sequenced, and protein productscan be produced. As used herein, the term also describes proteins thathave been produced following the transfer of genes into the cells ofplant host systems.

Selectable marker: a “selectable marker” is typically a gene that codesfor a protein that confers some kind of resistance to an antibiotic,herbicide or toxic compound, and is used to identify transformationevents. Examples of selectable markers include the streptomycinphosphotransferase (spt) gene encoding streptomycin resistance, thephosphomannose isomerase (pmi) gene that converts mannose-6-phosphateinto fructose-6 phosphate; the neomycin phosphotransferase (nptII) geneencoding kanamycin and geneticin resistance, the hygromycinphosphotransferase (hpt or aphiv) gene encoding resistance tohygromycin, acetolactate synthase (als) genes encoding resistance tosulfonylurea-type herbicides, genes coding for resistance to herbicideswhich act to inhibit the action of glutamine synthase such asphosphinothricin or basta (e.g., the bar gene), or other similar genesknown in the art.

Sense suppression: reduction in expression of an endogenous gene byexpression of one or more an additional copies of all or part of thatgene in transgenic plants.

T-DNA-Like: a “T-DNA-like” sequence is a nucleic acid that is isolatedfrom a selected plant species, or from a plant that is sexuallycompatible with the selected plant species, and which shares at least75%, 80%, 85%, 90%, or 95%, but not 100%, sequence identity withAgrobacterium species T-DNA. The T-DNA-like sequence may contain one ormore border or border-like sequences that are each capable ofintegrating a nucleotide sequence into another polynucleotide. A“P-DNA,” as used herein, is an example of a T-DNA-like sequence.

Trailer: Transcribed but not translated sequence following (or 3′ to) agene.

Transcribed DNA: DNA comprising both a gene and the untranslated leaderand trailer sequence that are associated with that gene, which istranscribed as a single mRNA by the action of the preceding promoter.

Transcription and translation terminators: the expression vectors of thepresent invention typically have a transcription termination region atthe opposite end from the transcription initiation regulatory region.The transcription termination region may be selected, for stability ofthe mRNA to enhance expression and/or for the addition ofpolyadenylation tails added to the gene transcription product (Alber &Kawasaki, Mol & Appl. Genetics 4: 19-34, 1982). Illustrativetranscription termination regions include the E9 sequence of the peaRBCS gene (Mogen et al., Mol. Cell Biol., 12: 5406-14, 1992) and thetermination signals of various ubiquitin genes.

Transformation of plant cells: a process by which DNA is stablyintegrated into the genome of a plant cell. “Stably” refers to thepermanent, or non-transient retention and/or expression of apolynucleotide in and by a cell genome. Thus, a stably integratedpolynucleotide is one that is a fixture within a transformed cell genomeand can be replicated and propagated through successive progeny of thecell or resultant transformed plant. Transformation may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion ofnucleic acid sequences into a prokaryotic or eukaryotic host cell,including Agrobacterium-mediated transformation protocols, viralinfection, whiskers, electroporation, heat shock, lipofection,polyethylene glycol treatment, micro-injection, and particlebombardment.

Transgene: a gene that will be inserted into a host genome, comprising aprotein coding region. In the context of the instant invention, theelements comprising the transgene are isolated from the host genome.

Transgenic plant: a genetically modified plant which contains at leastone transgene.

Using/Use of: The present invention envisions the use of nucleic acidfrom species other than that of the selected plant species to betransformed to facilitate the integration of native genetic elementsinto a selected plant genome, so long as such foreign nucleic acid isnot stably integrated into the same host plant genome. For instance, theplasmid, vector or cloning construct into which native genetic elementsare cloned, positioned or manipulated may be derived from a speciesdifferent to that from which the native genetic elements were derived.

Variant: a “variant,” as used herein, is understood to mean a nucleotideor amino acid sequence that deviates from the standard, or given,nucleotide or amino acid sequence of a particular gene or protein. Theterms, “isoform,” “isotype,” and “analog” also refer to “variant” formsof a nucleotide or an amino acid sequence. An amino acid sequence thatis altered by the addition, removal or substitution of one or more aminoacids, or a change in nucleotide sequence, may be considered a “variant”sequence. The variant may have “conservative” changes, wherein asubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. A variant may have“nonconservative” changes, e.g., replacement of a glycine with atryptophan. Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted may be foundusing computer programs well known in the art such as Vector NTI Suite(InforMax, MD) software.

It is understood that the present invention is not limited to theparticular methodology, protocols, vectors, and reagents, etc.,described herein, as these may vary. It is also to be understood thatthe terminology used herein is used for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention. It must be noted that as used herein and in theappended claims, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, a reference to “a gene” is a reference to one or more genes andincludes equivalents thereof known to those skilled in the art and soforth. Indeed, one skilled in the art can use the methods describedherein to express any native gene (known presently or subsequently) inplant host systems.

P-DNA Vectors

Agrobacterium-mediated transformation methods are the preferred means ofincorporating recombined DNA into plant cells. According to the presentinvention, a binary vector was developed to produce genetically modifiedpotato plants that contain only native potato nucleic acids. Such avector is different from conventional, Agrobacterium-mediatedtransformation vectors in three ways: (1) instead of anAgrobacterium-derived T-DNA sequence delineated by T-DNA borders, thepresent vector contains a native plant DNA (P-DNA) fragment that isflanked by border-like sequences, which support P-DNA transfer fromAgrobacterium to plant cells although they are structurally andfunctionally different from T-DNA borders, (2) the backbone of thepresent vector may contain a marker that, if integrated into the plantcell's genome, prevents these cells from developing into mature plants,and (3) the present vector does not contain a foreign selectable markergene between P-DNA termini.

The present invention demonstrates, surprisingly, that P-DNA fragmentsflanked by border-like sequences support DNA transfer from Agrobacteriuminto plant cells. P-DNA can be isolated from the genome of any plant byusing primers that are designed on the basis of homology between thetermini of a potato P-DNA and conventional T-DNA borders. Such fragmentscan then be tested and, if efficacious, used to transform that plantwith native DNA exclusively. It is also possible to search plant genomicdatabases for DNA fragments with regions that show homology with T-DNAborders by using programs such as ‘blastn’ (Altschul et al., J Mol Biol215: 403-10, 1990). The identified P-DNAs may then be modified toincrease their utility. For instance, internal fragments of the isolatedP-DNAs may be deleted and restriction sites may be added to facilitatecloning. It may also be efficacious to introduce point mutations at theterminal sequences to render the P-DNA more effective in transferringDNA.

Any gene expression cassette can be inserted between P-DNA border-likesequences. For potato transformations, such an expression cassette couldconsist of a potato promoter, operably linked to a potato gene and/or aleader or trailer sequence associated with that gene, and followed by apotato terminator. The expression cassette may contain additional potatogenetic elements such as a signal peptide sequence fused in frame to the5′-end of the gene, and a potato intron that could, for instance, beplaced between promoter and gene-of-interest to enhance expression. Fortransformation of wheat with a modified P-DNA, all genetic elements thatare inserted on the wheat P-DNA, including the P-DNA itself would bederived from wheat or plant species that are sexually compatible withwheat.

Another way to isolate P-DNAs is by generating a library ofAgrobacterium strains that contain random plant DNA fragments instead ofa T-DNA flanking a selectable marker gene. Explants infected with thislibrary can be placed on proliferation medium that contains anappropriate selectable agent to identify P-DNAs that support thetransfer of the marker gene from the vector in Agrobacterium to theplant cell.

It is possible that not just the native modified P-DNA, but alsoadditional plasmid sequences are co-transferred from Agrobacterium tothe plant cell during the transformation process. For the purposes ofthe present invention, this is an undesirable process because suchplasmid “backbone” sequences represent non-plant, foreign DNA, such asbacterial DNA. The present invention prevents transformed plant cellsthat contain backbone sequences from developing into mature plants.Thus, the present invention makes it possible to distinguishbackbone-containing and backbone-free transformation events during theregenerated shoot phase.

The method to select or screen against backbone integration eventsrelies on the presence of an expression cassette for a marker, such asthe isopentenyl phosphotransferase (IPT) gene, in the vector backbone,outside of the P-DNA. Upon backbone integration, the accumulation ofIPT-induced cytokinin will alter the shape of transformed shoots, andprevent these shoots to develop roots. Instead of the IPT gene, anyother gene that alters the shape, texture or color of the transformedplant's leaves, roots, stem, height or some other morphological featurecan be used to screen and/or select against backbone integration events.Such a gene is referred to herein as a “backbone integration marker.”Thus, the transformed plant that exhibits an altered morphologicalfeature attributable to the expression of the backbone integrationmarker gene is known to, contain in its genome foreign DNA in additionto the desired P-DNA. Accordingly, plants that exhibit a phenotypeassociated with the backbone integration marker are not desired.

The present invention is not limited to the use of only an IPT gene as abackbone integration marker; other genes can be used in such fashion.For example, a backbone integration marker may be an Agrobacteriumtranszeatine synthase (TZS) gene (Krall et al., FEBS Lett 527: 315-8,2002) or a recessive Arabidopsis gene hoc1 (Catterou et al., Plant J 30:273-87, 2002). This method can be more easily applied for use in thepresent invention than some methods that insert toxic genes in vectorbackbone sequences. See, for instance, EP 1 009,842.

By positioning a backbone integration marker gene, such as a functionalcytokinin gene upstream or downstream of the P-DNA, it isstraightforward to distinguish between transformation events.Transformed plants that exhibit an altered morphological feature arediscarded because they contain non-native DNA sequences integrated intothe genome.

Another strategy for identifying plants that are stably transformed withonly native DNA, is to employ the polymerase chain reaction. By usingprimers that are specifically designed to detect backbone sequences,plants can be identified and discarded that contain foreign backbonesequences in addition to the P-DNA. Other primer sets can subsequentlybe used to confirm the intact transfer of the P-DNA. Thus, by eitherusing the expression of a gene to change a morphological feature of aplant, or by screening for stably integrated foreign DNA in atransformed plant, plants stably transformed with only native DNAsequences can be identified and selected.

Genetic elements from a particular host plant can be inserted into theP-DNA sequence of a binary vector capable of replication in both E. coliand Agrobacterium. Introduction of the resulting vectors into disarmedAgrobacterium strains such as LBA4404 can be accomplished throughelectroporation, triparental mating or heat-shock treatment ofchemically competent cells. The new strains can then be used totransform individual plant cells through infection of whole plants orexplants.

Genetic elements from a particular host plant can be inserted into theP-DNA sequence of a binary vector capable of replication in both E. coliand Agrobacterium. Introduction of the resulting vectors intoAgrobacterium strains such as LBA4404 can be accomplished throughelectroporation, triparental mating or heat-shock treatment ofchemically competent cells. The new strains can then be used totransform individual plant cells through infection of whole plants orexplants. LBA4404 contains the disarmed Ti-plasmid pAL4404, whichcarries the virulence functions and a streptomycin resistance gene.

LifeSupport Vectors

Although the stable integration of bacterial marker genes into thegenomes of plant cells facilitates the identification of transformationevents, such modifications of plant genomes are undesirable becausemarker genes represent foreign DNA. Use of a foreign marker gene can beavoided by developing new Agrobacterium-based transformation methods.

One preferred embodiment is a novel method that relies on the use of twoAgrobacterium strains: one strain containing a binary vector with aselectable marker gene intended for transient expression in plantnuclei, and another strain carrying the P-DNA with the actual sequencesof interest intended for stable integration in plant genome (see Example7).

Upon co-infection with the Agrobacterium strains, some plant cells willreceive both a T-DNA with the marker gene and a P-DNA with the sequencesof interest. Instead of subsequently selecting for stable integration ofthe marker gene by subjecting the infected explants for a long period oftime to the appropriate antibiotic, explants are only briefly exposed tothe antibiotic. In this way, all plant cells that transiently expressthe marker gene will survive. Because T-DNAs will in most cases degradedue to endogenous nuclease activities rather than stably integrate intotheir host's genome, the majority of plant cells that survived thetransient selection are shown here to develop into shoots lacking amarker gene. The present invention, furthermore, demonstrates that asignificant proportion of these marker-free shoots contain stablyintegrated P-DNAs.

There are various tools to enhance the efficiency of marker-freetransformation. First, the present invention demonstrates that thisfrequency can be increased by sequentially infecting explants with twoAgrobacterium strains carrying the T-DNA/marker andP-DNA/sequences-of-interest, respectively. Explants are first infectedwith the P-DNA strain, and after about 4 to 6 hours with the T-DNAstrain.

Second, the T-DNA strain can be modified to express an omega-mutatedvirD2 gene. The modified virD2 protein will still support T-DNA transferto plant nuclei but limit the efficiency of a subsequent genomicintegration of T-DNAs (Shurvinton et al., Proc Natl Acad Sci USA, 89:11837-11841, 1992; Mysoreet al., Mol Plant Microbe Interact, 11:668-683, 1998). The most preferred method of expressing a modified virD2gene is by inserting an omega-mutated virD2 gene driven by the virDpromoter in the backbone of the T-DNA vector.

Third, stable T-DNA integration can be further impaired by insertingtelomere sequences close to the left- and right-border sequences of theT-DNA (Chiurazzi & Signer, Plant Mol. Biol., 26: 923-934,1994).

Fourth, the size of the T-DNA region carrying the marker gene can beincreased to enhance the frequency of T-DNAs and P-DNAs moving togetherinto the plant cell nucleus, and to reduce the frequency of genomicintegration of the T-DNA.

Fifth, the frequency of T-DNAs and P-DNAs moving together into the plantcell nucleus can also be enhanced by using a single Agrobacterium straincarrying two compatible binary vectors with the T-DNA and P-DNA,respectively. An example of two compatible binary vectors are a pSIM1301-derived vector and a pBI121-derived vector.

Because the transiently expressed marker gene will usually not integrateinto the plant genome, it is not necessary that both this gene and itsregulatory sequences represent native DNA. In fact, it may beadvantageous to use foreign regulatory sequences to promote high levelsof transient gene expression in infected plant cells. A surprisingdiscovery of the present invention is that an expression cassettecontaining the GUS gene followed by the terminator of the yeast alcoholdehydrogenase 1 (ADH1) was transiently expressed at high levels inpotato cells. A similar construct with the yeast CYC1 terminator,however, did not function adequately. It may also be possible to enhancetransient expression levels by operably linking a marker gene to anon-native promoter. Examples of such promoters are, e.g., syntheticpromoters such as glucocorticoid-inducible promoters (Mori et al., PlantJ., 27: 79-86, 2001; Bohner et al., Mol. Gen. Genet., 264: 860-70 2001),and non-native promoters such as the 35S promoters of cauliflower mosaicvirus and figwort mosaic virus, and fungal promoters.

As an alternative to the two-strain Agrobacterium-mediatedtransformation approach described above, plants may also be transformedwith a single strain that contains a P-DNA with both a native markergene and the actual sequences of interest. The present inventiondemonstrates that it is possible to use salt tolerance genes as nativemarkers for transformation. Such salt tolerance genes include crophomologs of the Arabidopsis genes SOS1 (Shi et al., Nat Biotechnol.2002), AtNHX1 (Apse et al., Science. 285: 1256-8, 1999), Avp1 (Gaxiolaet al., Proc Natl Acad Sci USA. 98: 11444-9, 2001), and CBF3 (Kasuga etal., Nat Biotechnol. 17: 287-91, 1999).

The rearrangements of genetic elements accomplished through theinventive Precise Breeding methodology could also occur spontaneouslythrough the process of genetic recombination. For instance, all plantscontain elements that can transpose from one to another chromosomallocation. By inserting into promoters or genes, such transposableelements can enhance, alter, and/or reduce gene expression. Forinstance, the AMu4 insertion of the maize Mutator element in thepromoter of the transcriptional regulator gene P-wr causes stripy redpericarps. Insertion of the same element in the promoter of theleaf-specific MADS-box gene ZMM19 resulted in expression of this gene inthe inflorescences of maize, causing a foliaceous elongation of theglumes and other changes in male and female inflorescences, resulting inthe famous phenotype of pod corn. Because of its bizarre tassels andears, pod corn was of religious significance for certain native Americantribes. Many genes are also rearranged through other transposon-inducedmodifications such as inversions, deletions, additions, and ectopicrecombinations (Bennetzen, Plant Mol Biol 42: 251-69, 2000).Furthermore, plant DNA rearrangements frequently occur through theprocess of intragenic recombination. For instance, by recombining genesinvolved in resistance against specific pathogens, plants are able todevelop resistance genes with new specificities and, thus, co-evolvewith their pathogens (Ellis et al., Trends Plant Sci 5: 373-9, 2000).Another example of intragenic recombination relates to how plantsreproduce: plants transition from cross-fertilizing to self-fertilizingby recombining genes involved in self-incompatibility (Kusaba et al.,Plant Cell 13: 627-43, 2001). Other processes that promote genomeevolution include, for instance, chromosome breakage andinterchromosomal recombination.

Enhancing the Nutritional Value of Plants and Food Crops

To modify negative traits such as acrylamide accumulation duringprocessing, glycoalkaloid accumulation, accumulation of undesirableadvanced glycation products, CIPC accumulation, low levels of resistantstarch, bruise susceptibility, cold-induced sweetening, diseasesusceptibility, low yield and low quality in crop plants through precisebreeding, at least one specific expression cassette is incorporated intoa host genome. Three different methods are used to eliminate negativetraits: (1) overexpression of genes that prevent the occurrence ofnegative traits, (2) overexpression of mutated versions of genesassociated with negative traits in order to titrate out the wild-typegene products with non-functional proteins, and (3) silencing specificgenes that are associated with a negative trait by expressing at leastone copy of a leader or trailer fragment associated with that gene inthe sense and/or antisense orientation.

One example of an endogenous gene that is associated with a negativetrait in potato and can be modified in vitro so that it encodes anon-functional protein is the polyphenol oxidase (PPO) gene. Upon impactinjury, the PPO gene product is released from the plastid into thecytoplasm (Koussevitzky et al., J. Biol. Chem., 273: 27064-9, 1998),where it will mediate the oxidation of phenols to create a variety ofphenoxyl radicals and quinoid derivatives, which are toxic and/orultimately form undesirable polymers that leave dark discolorations, or“black spots” in the crop.

Overexpressing a mutant PPO gene that contains a non-functionalcopper-binding domain can lower the activity of all PPO genes that aremainly expressed in tubers and associated organs such as sprouts. Themutations render the polyphenol oxidase protein inactive because it isunable to bind copper. The skilled artisan would know where to makepoint mutations that would, in this case, compromise the function of agene product. The applicants identified the copper binding domain inpotato PPO by aligning the potato PPO protein sequence with a sweetpotato PPO protein sequence (Klabunde et al., Nat Struct. Biol., 5:1084-90, 1998). Areas of conservation, particularly those containingconserved histidine residues in copper-binding sites, were targets forinactivating the transgene product. Because the almost complete absenceof PPO activity in such organs may negatively impact the plant's abilityto resist pathogens, the present invention also describes an improvedmethod of only lowering a specific PPO gene that is predominantlyexpressed in all parts of the mature tuber except for the epidermis.Silencing of this specific PPO gene by using a trailer sequenceassociated with that gene does not reduce PPO expression in the tuberepidermis, the part of the tuber that is most directly exposed topathogens attempting to infect.

Enzymatic browning induced by the PPO gene not only reduces the qualityof potato tubers; it also negatively affects crop foods such as wheat,avocado, banana, lettuce, apple, and pears.

Other genes that are associated with negative traits and can be silencedby using the leader or trailer sequences associated with those genesinclude the potato R1 gene and L-type phosphorylase genes. Both genesare involved in the degradation of starch to reducing sugars, such asglucose and fructose, which upon heating participate in the Maillardreaction to produce toxic products such as acrylamide. The presentinvention demonstrates that a reduction of cold-induced sweetening bylowering R1 or phosphorylase activity leads to a reduction of bothnon-enzymatic browning and acrylamide accumulation during the fryingprocess of potatoes.

The invention also demonstrates the utility of overexpressing certainnative genes in genetically modified crops. Levels of Maillard-reactionproducts such as acrylamide were reduced significantly by lowering theconversion of sucrose to reducing sugars through overexpression of anewly isolated vacuolar invertase inhibitor gene in potato.

The present invention also predicts that potato tubers displaying eitheran increased level of invertase inhibitor expression or a reduced levelof R1 or phosphorylase expression will not require the intensivetreatment with chemical sprout inhibitors such as CIPC prior to storagebecause their lowered levels of reducing sugars will (1) delaysprouting, and (2) allow storage at lower temperatures, thus furtherdelaying sprouting. The highly reduced CIPC-residue levels, or theabsence thereof, further enhances the nutritional value of processedfoods derived from plants containing certain modified P-DNAs describedhere.

Thus, French fries or chips derived from tubers that contain themodified P-DNA will contain strongly reduced CIPC residue levels,further boosting their nutritional value.

The effect of simultaneously downregulating the expression of the PPOand either R1 or phosphorylase genes in potato tubers is synergisticbecause reducing sugars are not only required for non-enzymatic browningthrough the Maillard reaction but also for browning mediated by the PPOenzyme. Decreased levels of reducing sugars in transgenic potato tuberswill, therefore, also limit PPO activity and black spot bruisesusceptibility. Thus, PPO, R1, and phosphorylase genes, and/or theleader or trailer sequences that are associated with these genes,represent DNA segments of interest that can be isolated, modified andreintroduced back into the plant to down-regulate the expression ofthese genes.

Apart from developing bruise resistance and reduced cold-inducedsweetening, there are many other traits that can be introduced throughPrecise Breeding without using foreign DNA. For instance, diseaseresistance genes can be isolated from wild potato species and insertedinto the genomes of disease susceptible varieties.

The Environmental Benefits of Modifted Plants and Crops

As described above, reduced levels of either R1 orphosphorylase resultin a reduced phosphorylation of starch. This reduction in starchphosphorylation results in a 90% decrease in phosphate content of potatotubers (Vikso-Nielsen, Biomacromolecules, 2: 836-43, 2001). This willresult in a reduction in phosphate levels in wastewaters from potatoprocessing plants, which are currently about 25-40 mg/L. Thus, the useof low-phosphate tubers will reduce the release of phosphates into theenvironment and help to protect important ecosystems. Furthermore,low-phosphate potatoes may require less phosphate fertilization foroptimal growth and yield, which would support a more sustainableagriculture by delaying the depletion of available phosphate resources.

Enhancing the Agricultural Performance of Plants and Food Crops

Apart from reduced bruise susceptibility and reduced cold-sweetening,which are two important processing traits, the present invention alsoprovides salt tolerance, an increasingly important input trait. Some ofthe modified P-DNA constructs described in the present invention containa salt tolerance gene as native marker for transformation. Importantly,the utility of this gene is not limited to a screening step in thetransformation procedure. Overexpression of the salt tolerance gene inpotato plants reduces stress symptoms induced by high salinity soillevels, and will make it possible to grow new varieties containing amodified P-DNA on a growing percentage of agricultural lands thatcontain salinity levels exceeding the maximum 2 millimhos/cm electricalconductivity levels that are optimal for growing conventional varieties.

Using Regulatory Elements Isolated from a Selected Plant Species or froma Species Sexually Compatible with the Selected Plant Species

Once the leader, gene or trailer has been isolated from the plantspecies of interest, and optionally modified, it can be operably linkedto a plant promoter or similar regulatory element for appropriateexpression in plants. Regulatory elements such as these serve to expressuntranslated sequences associated with a gene of interest in specifictissues or at certain levels or at particular times.

Dependent on the strategy involved in modifying the trait, it may benecessary to limit silencing to a particular region of the plant. Thepromoter normally driving the expression of the endogenous gene may notbe suitable for tissue-specific expression. As described in the sectionabove, stable integration of bacterial or viral regulatory components,such as the cauliflower mosaic virus 35S “super” promoter, can result inunpredictable and undesirable events. Thus, one aspect of the presentinvention uses promoters that are isolated from the selected host plantspecies.

In a preferred embodiment of the instant invention, for use in S.tuberosum, the leader or trailer sequences associated with R1,phosphorylase, and PPO genes are operably linked to the granule-boundstarch synthase gene promoter (Rohde et al., J Gen & Breed, 44, 311-315,1990). This promoter has been used frequently by others to drive geneexpression and is particularly active in potato tubers (van der Steegeet al., Plant Mol Biol, 20: 19-30, 1992; Beaujean et al., Biotechnol.Bioeng, 70: 9-16, 2000; Oxenboll et al., Proc Natl Acad Sci USA, 9:7639-44, 2000). This promoter may also be used, in a preferredembodiment, for expression of the modified leader or trailer sequencesof R1, phosphorylase, and PPO genes.

Alternatively, other potato promoters can be operably linked tosequences of interest from potato. Such promoters include the patatingene promoter (Bevan et al., Nucleic Acids Res, 14: 4625-38, 1986), or afragment thereof, that promotes expression in potato tubers, the potatoUDP-glucose pyrophosphorylase gene promoter (U.S. Pat. No. 5,932,783)and the promoter of the ubiquitin gene (Garbarino et al., Plant Physiol,109: 1371-8, 1995).

The transcription of leaders and/or trailers can also be regulated byusing inducible promoters and regulatory regions that are operablylinked in a construct to a polynucleotide of interest. Examples ofinducible promoters include those that are sensitive to temperature,such as heat or cold shock promoters. For instance, the potato ci21A-,and C17-promoters are cold-inducible (Kirch et al., Plant Mol. Biol, 33:897-909, 1997; Schneider et al., Plant Physiol, 113: 335-45, 1997).

Other inducible promoters may be used that are responsive to certainsubstrates like antibiotics, other chemical substances, or pH. Forinstance, abscisic acid and gibberellic acid are known to affect theintracellular pH of plant cells and in so doing, regulate the Rab 16Agene and the alpha-amylase 1/6-4 promoter (Heimovaara-Dijkstra et al.,Plant Mol Biol, 4 815-20, 1995). Abscisic acid, wounding and methyljasmonate are also known to induce the potato pin2 promoter (Lorberth etal., Plant J, 2: 477-86, 1992).

In another example, some nucleotide sequences are under temporalregulation and are activated to express a downstream sequence onlyduring a certain developmental stage of the plant or during certainhours of the day. For instance, the potato promoter of the small subunitof ribulose-1,5-bisphosphate carboxylase (rbcS) gene can directcell-specific, light-regulated expression (Fritz et al., Proc Natl AcadSci USA, 88: 4458-62, 1991). The skilled artisan is well versed in theseexemplary forms of inducible promoters and regulatory sequences.

The use of certain polyadenylation signals may also be useful inregulating expression, by varying the stability of the mRNA transcript.In particular, some polyadenylation signals when operably linked to the'3 end of a polynucleotide cause the mRNA transcript to becomeaccessible to degradation.

Thus, it is possible to regulate expression of a gene by operablylinking it with one or more of such promoters, regulatory sequences, 3′polyadenylation signals, 3′ untranslated regions, signal peptides andthe like. According to the instant invention, DNA sequences andregulatory elements such as those described herein, and which willultimately be integrated into a plant genome, are obtained from DNA ofthe selected plant species to be modified by the Precise Breedingprocess of the present invention. That is, DNA sequences and regulatoryelements that are derived, isolated and cloned from other species, suchas from bacteria, viruses, microorganisms, mammals, birds, reptiles andsexually incompatible plant species are not integrated into the genomeof the transformed plant. DNA foreign to the selected plant speciesgenome may be used in the present invention to create a transformationconstruct, so long as that foreign DNA is not integrated into a plantgenome.

Not only does the present invention provide a method for transforming aplant species by integrating DNA obtained from the selected plantspecies, or from a plant that is sexually-compatible with the selectedplant species, it also provides a means by which the expression of thatDNA can be regulated. Accordingly, it is possible to optimize theexpression of a certain sequence, either by tissue-specific or someother strategy, as previously described.

Using 3′ Terminator Sequences Isolated from a Selected Plant Species

In addition to regulatory elements that initiate transcription, thenative expression cassette also requires elements that terminatetranscription at the 3′-end from the transcription initiation regulatoryregion. The transcription termination region and the transcriptioninitiation region may be obtained from the same gene or from differentgenes. The transcription termination region may be selected,particularly for stability of the mRNA to enhance expression.

This particular element, the so-called “3′-untranslated region” isimportant in transporting, stabilizing, localizing and terminating thegene transcript. In this respect, it is well known to those in the art,that the 3′-untranslated region can form certain hairpin loop.Accordingly, the present invention envisions the possibility of operablylinking a 3′ untranslated region to the 3′ end of a clonedpolynucleotide such that the resultant mRNA transcript may be exposed tofactors which act upon sequences and structures conferred by the 3′untranslated region.

A 3′ sequence of the ubiquitin gene can be subcloned from the plantspecies from which the promoter and transgene were isolated and inserteddownstream from a transgene to ensure appropriate termination oftranscription. Both exemplary transgenes can be fused to the termninatorsequence of the potato Ubiquitin gene (Ubi3) regardless of whichpromoter is used to drive their expression.

EXAMPLES Example 1 Cloning of P-DNAs

This example demonstrates that T-DNA borders are specific toAgrobacterium. It also shows that plants contain T-DNA border-likesequences, and it provides the sequence of DNA fragments isolated frompotato and wheat that are delineated by such border-like sequences.

Conventional transformation systems use Agrobacterium-derived T-DNAs asvehicles for the transfer of foreign DNA from Agrobacterium to plantcells (Schilperoort et al., U.S. Pat. No. 4,940,838, 1990). AlthoughT-DNAs usually comprise several hundreds of basepairs, delineated by aleft-border (LB) and right-border (RB) repeat, they can also merelyconsist of such borders. The T-DNA borders play an essential role in theDNA transfer process because they function as specific recognition sitesfor virD2-catalyzed nicking reaction. The released single stranded DNA,complexed with Agrobacterial virD2 and virE2, is transferred to plantcell nuclei where it often integrates successfully into the plantgenome. All T-DNA borders that have been used for foreign DNA transferare derived from nopaline and octopine strains of Agrobacteriumtumefaciens and A. rhizogenes (Table 2). These borders and often someflanking Agrobacterium DNA are present in thousands of binary vectorsincluding, for example, pPAM (AY027531), pJawohl (AF408413), pYL156(AF406991), pINDEX (AF294982), pC1300 (AF294978), pBI121 (AF485783),pLH9000 (AF458478), pAC161 (AJ315956), BinHygTOp (Z37515), pHELLSGATE(AJ311874), pBAR-35S (AJ251014), pGreen (AJ007829), pBIN19 (X77672),pCAMBIA (AF354046), pX6-GFP (AF330636), pER8 (AF309825), pBI101(U12639), pSKI074 (AF218466), pAJ1 (AC138659), pAC161 (AJ315956),pSLJ8313 (Y18556), and pGV4939 (AY147202). Recently, two homologs ofT-DNA borders were identified in the chrysopine-type Ti plasmid pTiChry5(Palanichelvam et al., Mol Plant Microbe Interact 13: 1081-91, 2000).The left border homolog is identical to an inactive border homologlocated in the middle of the T-DNA of pTi15955. The right border homologis unusually divergent from the sequence of functional T-DNA borders. Itis therefore unlikely that these homologs are functionally active insupporting DNA transfer from pTiChry5 to plant cells.

Development of a new method that makes it possible to transform plantswith only native DNA requires, in the first place, a replacement of theT-DNA including LB and RB. Unfortunately, advanced BLAST searches ofpublic databases including those maintained by The National Center ForBiotechnology Information, The Institute for Genomic Research, andSANGER failed to identify any border sequences in plants. It wastherefore necessary to consider plant DNA sequences that are similar butnot identical to T-DNA borders, designated here as “border-like”(border-like). Examples of plant border-like sequences that wereidentified in public databases are shown in Table 2. The challenge intrying to replace T-DNA borders with border-like sequences is thatborder sequences are highly conserved (see Table 2). A large part ofthese sequences is also highly conserved in the nick regions of otherbacterial DNA transfer systems such as that of IncP, PC194, and φX174,indicating that these sequences are essential for conjugative-like DNAtransfer (Waters et al., Proc Natl Acad Sci 88: 1456-60, 1991). Becausethere are no reliable data on border sequence requirements, the entireborder seems therefore important in the nicking process. A single studythat attempted to address this issue by testing the efficacy of bordermutants in supporting DNA transfer is unreliable because negativecontrols did not appear to function appropriately (van Haaren et al.,Plant Mol Biol 13: 523-531, 1989). Furthermore, none of the results ofthis study were confirmed molecularly. Despite these concerns, twopossibly effective border mutants are shown in Table 2 as well.

Based on the homology among border sequences, a T-DNA border motif wasidentified (Table 2). Although this motif comprises 13,824 variants,many of which may not function—or may be inadequate—in transferring DNA,it represents the broadest possible definition of what a T-DNA bordersequence is or may be. This border motif was then used to searchpublicly available DNA databases for homologs using the “Motif Alignmentand Search Tool” (Bailey and Gribskov, Bioinformatics 14: 48-54, 1998)and “advanced BLASTN” (“penalty for nucleotide mismatch”=−1;“expect”=10⁵; Altschul et al., Nucleic Acids Res 25: 3389-3402, 1997).Again, these searches did not identify any identical matches inorganisms other than Agrobacterium.

To try and increase the chance of isolating a potato DNA fragmentcontaining border-like sequences that correspond to the border motif,DNA was isolated from 100 genetically diverse accessions (the so-called“core collection,” provided by the US Potato Genebank, WI). This DNA waspooled and used as template for polymerase chain reactions using avariety of oligonucleotides designed to anneal to borders or border-likesequences. Amplified fragments were sequence analyzed, and the sequencewas then confirmed using inverse PCR with nested primers. One of thepotato DNA fragments that was of particular interest contains a novelsequence without any major open reading frames that is delineated byborder-like sequences (Table 2). One of the border-like sequences ofthis fragment contains at least 5 mismatches with T-DNA borders; theother border-like sequence contains at least 2 mismatches. Although bothsequences contain one mismatch with the border motif, they were testedfor their ability to support DNA transfer. For that purpose, thefragment was first reduced in size to 0.4-kilo basepairs by carrying outan internal deletion (SEQ ID NO.: 1). The resulting fragment wasdesignated “P-DNA” (plant DNA) to distinguish it from theAgrobacterium-derived T-DNA. A similar fragment was isolated from thegenome of the potato variety Russet Ranger, but has not been used forany further experiments.

Based on the divergence between P-DNA and T-DNA borders, the elongaseamplification system (Life Technologies) was used with the followingdegenerate primers to isolate a P-DNA from wheat:5′-GTTTACANHNBNATATATCCTGYCA-3′ (Bor-F) (SEQ ID NO. 56), and5′-TGRCAGGATATATNVNDNTGTAAAC-3′ (Bor-R) (SEQ ID NO. 57). The resulting825-bp fragment is shown in SEQ ID NO.: 2, and was used to replace theT-DNA of a conventional binary vector. The efficacy of this constructcan be tested by inserting an expression cassette for the GUS genebetween P-DNA termini, and infecting wheat with an Agrobacterium straincarrying the resulting vector.

Example 2 Tobacco Transformation with P-DNA Vectors

This Example demonstrates that, despite structural (sequence divergence)and functional (transformation frequencies) differences between P-DNAtermini and T-DNA borders, a P-DNA can be used in a similar way as aT-DNA to transfer DNA from Agrobacterium to tobacco cells.

A T-DNA-free vector that can be maintained in both E. coli and A.tumefaciens was obtained by removing the entire T-DNA region of theconventional binary vector pCAMBIA1301 (Cambia, AU). This wasaccomplished by simultaneously ligating a 5.9 kb SacII—SphI fragment ofpSIM1301 with 2 fragments amplified from pCAMBIA1301 using theoligonucleotides pairs: 5′-CCGCGGTGATCACAGGCAGCAAC-3′ (SEQ ID NO. 58)and 5′-AAGCTTCCAGCCAGCCAACAGCTCCCCGAC-3′ (SEQ ID NO. 59), and5′-AAGCTTGGCTACTAGTGCGAGATCTCTAAGAGAAAAGAGCGTTTA-3′ (SEQ ID NO. 60), and5′-GCATGCTCGAGATAGGTGACCACATACAAATGGACGAACGG-3′ (SEQ ID NO. 61),respectively.

To make it possible to screen against backbone integration events, anexpression cassette comprising the Agrobacterium isopentenyl transferase(IPT) gene driven by the Ubi3 promoter and followed by the Ubi3terminator (SEQ ID NO.: 3) was inserted as 2.6 kbp SacII fragment intothe backbone of the T-DNA-free vector described above, yieldingpSIM100-OD-IPT. Transformed plant cells expressing the IPT gene areexpected to accumulate cytokinins and grow into abnormal shoots thatcannot develop roots.

The 0.4 kb P-DNA fragment described in Example 1 was inserted intopSIM100-OD-IPT to generate pSIM111 (FIG. 1; SEQ ID NO.: 4).

To test whether pSIM111 can be used to obtain transformed plantscarrying P-DNAs (including any sequences located between P-DNA termini)without the additional vector backbone, a neomycin phosphotransferase(NPTII) gene expression cassette was inserted into the P-DNA of pSIM111to create pSIM108 (FIG. 1).

The efficacy of P-DNA termini in supporting DNA transfer was tested bycomparing transformation frequencies between pSIM108 and a controlvector that contained a modified P-DNA with conventional T-DNA borders.This control vector, designated pSIM109, was generated by amplificationof the entire P-DNA containing the NPTII gene expression cassette withthe oligonucleotide pairs: 5′-ACTAGTGTTTACCCGCCAATATATCCTGTCAGAG-3′ (SEQID NO. 62), and 5′-AAGCTTTGGCAGGATATATTGTGGTGTAAACGAAG-3′ (SEQ ID NO.63). A second control vector that was used for these experiments is theconventional binary vector pBI121 (Genbank accession number AF485783),which contains the same NPTII expression cassette inserted on a regularT-DNA. The binary vectors were introduced into Agrobacterium tumefaciensLBA4404 cells as follows. Competent LB4404 cells (50 uL) were incubatedfor 5 minutes at 37° C. in the presence of 1 μg of vector DNA, frozenfor about 15 seconds in liquid nitrogen (about −196° C.), and incubatedagain at 37° C. for 5 minutes. After adding 1 mL of liquid broth (LB),the treated cells were grown for 3 hours at 28° C. and plated on LB/agarcontaining streptomycin (100 mg/L) and kanamycin (100 mg/L). The vectorDNAs were then isolated from overnight cultures of individual LBA4404colonies and examined by restriction analysis to confirm the presence ofintact plasmid DNA.

Test transformations of the model plant tobacco were carried out bygrowing a 10-fold dilution of overnight-grown LBA4404::pSIM108 cells for5-6 hours, precipitating the cells for 15 minutes at 2,800 RPM, washingthem with MS liquid medium (Phytotechnology) supplemented with sucrose(3%, pH 5.7) and resuspending the cells in the same medium to anOD_(600nm) of 0.2. The suspension was then used to infect leaf explantsof 4-week-old in vitro grown Nicotiana tabacum plants. Infected tobaccoexplants were incubated for 2 days on co-culture medium (1/10 MS salts,3% sucrose, pH 5.7) containing 6 g/L agar at 25° C. in a Percival growthchamber (16 hrs light) and subsequently transferred to M401/agar mediumcontaining timentine (150 mg/L) and kanamycin (100 mg/L). The number ofcalli per explant that developed within the next 4 weeks is shown inTable 3. Our data demonstrate that P-DNAs delineated by either nativetermini or conventional T-DNA borders are about 50% more effective intransforming tobacco than T-DNAs. The increased efficiency of P-DNAtransfer may be due to either its different CG content or other unknownstructural features of the P-DNA.

Example 3 Potato Transformation with P-DNA Vectors

This Example demonstrates that a P-DNA can be used in a similar way as aT-DNA to transfer DNA from Agrobacterium to potato cells.

Potato transformations were carried out by infecting stem explants of4-week-old in vitro grown Russet Ranger plantlets with Agrobacteriumstrains according to the following procedure. Ten-fold dilutions ofovernight-grown cultures were grown for 5-6 hours, precipitated for 15minutes at 2,800 RPM, washed with MS liquid medium (Phytotechnology)supplemented with sucrose (3%, pH 5.7), and resuspended in the samemedium to an OD_(600nm) of 0.2. The resuspended cells were then used toinfect 0.4-0.6 mm internodal potato segments. Infected stems wereincubated for 2 days on co-culture medium (1/10 MS salts, 3% sucrose, pH5.7) containing 6 g/L agar at 22° C. in a Percival growth chamber (16hrs light) and subsequently transferred to callus induction medium (CIM,MS medium supplemented with 3% sucrose 3, 2.5 mg/L of zeatin riboside,0.1 mg/L of naphthalene acetic acid, and 6 g/L of agar) containingtimentine (150 mg/L) and kanamycin (100 mg/L). After 1 month of cultureon CIM, explants were transferred to shoot induction medium (SIM, MSmedium supplemented with 3% sucrose, 2.5 mg/L of zeatin riboside, 0.3mg/L of giberelic acid GA3, and 6 g/L of agar) containing timentine andkanamycin (150 and 100 mg/L respectively). After 3-4 weeks, the numberof explants developing transgenic calli and/or shooting was counted. Asshown in tobaco, the number of stem explants infected with pSIM108 thatshowed calli was higher than those in control experiments with theconventional binary vector pBI121 (Table 3). Shoots that subsequentlyarose from these calli could be grouped into two different classes. Thefirst class of shoots was phenotypically indistinguishable from controlshoots transformed with LBA::pBI121. The second class of shootsdisplayed an IPT phenotype. Shoots of the latter class were stunted ingrowth, contained only very small leaves, displayed a light-green toyellow color, and were unable to root upon transfer to hormone-freemedia. To confirm that shoots with an IPT phenotype contained the IPTgene stably integrated in their genomes, all shoots were transferred toMagenta boxes containing MS medium supplemented with 3% sucrose andtimentine 150 mg/L, allowed to grow for 3 to 4 additional weeks, andused to isolate DNA. This plant DNA served as template in PCR reactionswith an oligonucleotide pair designed to anneal to the IPT gene: 5′-GTCCAA CTT GCA CAG GAA AGA C-3′, and 5′-CAT GGA TGA AAT ACT CCT GAG C-3′.As shown in Table 4, the PCR experiment confirmed a strict correlationbetween IPT phenotype and presence of the IPT gene. The presence ofbackbone DNA was also examined in plants obtained from a transformationwith pBII21. This was done by performing PCR reactions on DNA isolatedfrom the transformation events with the ‘pBI121 backbone primers’:5′-CGGTGTAAGTGAACTGCAGTTGCCATG-3′ (SEQ ID NO. 64), and5′-CATCGGCCTCACTCATGAGCAGATTG-3′ (SEQ ID NO. 65). Amplification of a 0.7kbp band is indicative for backbone integration. By comparing the datapresented in Table 4, it can be concluded that backbone integrationfrequencies are similar for P-DNA vectors and T-DNA vectors.

A second PCR experiment was carried out to test whether IPT-free plantsdid not contain any other backbone sequences. Because the IPT expressioncassette is positioned close to the left border-like sequences, theoligonucleotide pair for this experiment was designed to anneal tobackbone sequences close to the right border-like sequence:5′-CACGCTAAGTGCCGGCCGTCCGAG-3′ (SEQ ID NO. 66), and5′-TCCTAATCGACGGCGCACCGGCTG-3′ (SEQ ID NO. 67). Data from thisexperiment confirm that plants that are positive for the IPT gene arealso positive for this other part of the backbone.

Similar experiments were carried out with the potato variety RussetBurbank. Based on an assessment of IPT phenotypes, the backboneintegration frequencies for pSIM108 and pSIM109 were shown to becomparable to those in Russet Ranger (see Tables 4 and 5).

Example 4 Potato Invertase Inhibitor Gene

Using conventional transformation methods, this Example demonstratesthat overexpressing a novel potato invertase inhibitor gene enhances theprocessing and health characteristics of potato tubers.

The following primers were designed to amplify a new potato homolog ofthe tobacco vacuolar invertase inhibitor Nt-inhh1 (Greiner et al.,Nature Biotechnology, 17, 708-711, 1999):5′-AAAGTTGAATTCAAATGAGAAATTTATTC-3′ (SEQ ID NO. 68), and5′-TTTTAAGCTTTCATAATAACATTCTAAT-3′ (SEQ ID NO. 69). The amplificationreaction was performed by mixing the following components: 4 μl plantDNA, 2 μl forward primer (10 pM/ml), 2 μl reverse primer, 25 μl HotStart Master Mix (Qiagen Catalog Nr. 203443), and 17 μl water. Thisreaction mix was subjected to the following polymerase chain reaction(PCR) conditions using a PTC-100 thermocycler (M J Research): (1) 5minutes at 95° C. (1 cycle), (2) 1 minute at 94° C., 1 minute at 45° C.and 4 minutes at 72° C. (35 cycles), and (3) 10 minutes at 72° C. (1cycle). The total product was loaded on a 0.8% agarose gel, and a 540base pair band was purified from gel using QIAquick Gel Extraction Kit(Qiagen, CA). This purified fragment was then ligated into pGEM-T Easy(Promega, WI) and transformed into E. coli DH5-alpha using MaxEfficiency Competent Cells (GibcoBRL, MD). Sequence analysis ofrecombinant plasmid DNA isolated from transformed DH5-alpha revealed thepresence of a single open reading frame consisting of 543 base pairsthat encodes for a putative 181-amino acid protein (SEQ ID NO.: 5);clustal-aligment revealed 70% homology to Nt-inhh (FIG. 2). This highlevel of homology extends to the 15-amino acid N-terminal domain,indicating that the potato homolog is targeted to the vacuole.Interestingly, the potato invertase inhibitor homolog, designatedSt-inh1, shares only 43% homology with the patented tobacco cell wallinvertase inhibitor designated Nt-inh1 (Patent WO98/04722; FIG. 2).

Although the St-inh1 gene is present in unmodified potato tubers, itsexpression level is inadequate for full inhibition of invertase andreduced cold-induced sweetening. To increase the storage characteristicsof potato, the St-inh1 gene was fused to a new tuber-enhanced promoterof the granule-bound starch synthase (GBSS) gene, which is known topromote high levels of gene expression in tubers. The GBSS promoter wasisolated from the potato cultivar Russet Ranger by carrying out a PCRreaction using the forward primer 5′-GAACCATGCATCTCAATC-3′ (SEQ ID NO.70) and the reverse primer 5′-GTCAGGATCCCTACCAAGCTACAGATGAAC-3′ (SEQ IDNO. 71). Sequence analysis of the amplified product cloned in pGEM-Tdemonstrated that this new promoter contains 658 basepairs (SEQ ID NO.:6). The resulting promoter/gene fusion was then ligated to the 3′regulatory sequence of the potato ubiquitin gene (UbiT; SEQ ID NO.: 7),thus ensuring appropriate termination of transcription of the invertaseinhibitor gene.

This expression cassette was inserted between T-DNA borders of a binaryvector, and the resulting vector pSIM320 was used to transform RussetRanger as described above. Three cuttings of nine independent transgeniclines were planted in soil and grown for four weeks in a growth chamber(11 hrs light; 20° C.). At least 3 minitubers were then harvested fromeach line and transferred to a refrigerator set at 4° C. to inducecold-sweetening. After 4 weeks, the glucose levels in these cold-storedminitubers were determined by using either an Accu-Chek meter and teststrips (Roche Diagnostics, IN) or a glucose oxidase/peroxidase reagent(Megazyme, Ireland). These levels were compared with the average glucoselevels in both 6 untransformed lines and 6 “vector control” linestransformed with a pSIM110-derived vector lacking the invertaseinhibitor gene. As shown in Table 6, three transgenic lines accumulatedless than 40% of the glucose in “vector control” lines demonstratingthat the potato invertase inhibitor homolog is functionally active.

The following experiment showed that the amount of reducing sugarspresent in tubers correlates with acrylamide production during tuberprocessing. Russet Ranger potato tubers were freshly harvested from thefield and stored at 4° C. to induce cold-sweetening; control tubers werestored at 18° C. After 4 weeks, glucose levels were determined in bothgroups of tubers. Subsequently, tubers were washed, blanched for either8 minutes or 12 minutes at 165° F., cut into 0.290×0.290 shoestringstrips, dipped in a 1% sodium acid pyrophosphate solution at 160° F.,dried at 160° F. until 14±2% dryer weight loss is achieved, fried at390° F. for 40 seconds to attain 64±2% first fry moisture, and frozenfor 20 minutes at −15° F., shaking the tray 2-3 times in the first 6minutes. The resulting French fries were then analyzed for acrylamidelevels by Covance laboratory (WI). As shown in Table 7, the glucoselevels in tubers stored at 18° C. were below the detection level of 0.1mg/g whereas cold-stored tubers contained on average 3.4 mg/g glucose.This table also shows that fries produced from the latter potatoescontain about 10-fold higher levels of acrylamide than fries producedfrom potatoes stored at 18° C. Even by using a shorter blanch time for18° C.-stored potatoes than for 4° C.-stored potatoes to produce frieswith a similar color (color ids of 78 and 71, respectively), a 5-folddifference in acrylamide accumulation was obtained (Table 7). Thus,there appears to be a straight correlation between the amount ofreducing sugars such as glucose in tubers and the accumulation ofacrylamide in fries derived from these tubers.

To determine whether the reduced glucose levels in pSIM320 lines wouldlimit the processing-induced accumulation of acrylamide, cold-storedpSIM320 minitubers were processed by cutting into wedges, blanching for8 minutes, dipping in 0.5% SAPP for 30 seconds, drying for 4.5 minutesat 160° F., frying for 40 seconds at 380° F., freezing for 15 minutes at−15° F., and finally drying for 3 minutes and 10 seconds at 160° F. Theprocessed material was then shipped to Covance laboratory for acrylamidedeterminations. As shown in Table 6, French fries obtained fromminitubers with the lowest amounts of glucose accumulated the lowestlevels of acrylamide. A 40% reduction in glucose levels in lines “320-2”and “320-4” is associated with a 5-fold reduction in acrylamide levels.

Example 5 Leader and Trailer Sequences Associated with the Potato R1Gene

Using conventional transformation methods, this Example demonstratesthat a novel leader sequence associated with the potato R1 gene can beused effectively to enhance the processing and health characteristics ofpotato tubers. It also predicts that a novel trailer associated withthat same gene can be exploited in the same way.

As an alternative to overexpressing the invertase inhibitor gene,methods were developed to limit acrylamide production without using anyactual gene sequences. One such method is based on silencing thetuber-expressed R1 gene. Previously, it was shown that thisstarch-related gene can be silenced through antisense expression of a1.9-kb gene fragment derived from that gene (Kossmann et al., U.S. Pat.No. 6,207,880). However, the antisense expression of large DNA fragmentsis undesirable because such fragments contain new open reading frames(Table 1). As a safer approach to the one described above, a smallleader sequence associated with the R1 gene was isolated from potato.This leader was obtained by performing a rapid amplification of cDNAends with the 5′ RACE kit supplied by GIBCO BRL on total RNA from thetubers of Russet Ranger potato plants. Sequence analysis demonstratedthat the R1-associated leader consists of 179 basepairs (SEQ ID NO.: 8).Both a sense and antisense copy of this leader sequence, separated bythe potato Ubiquitin intron (SEQ ID NO.: 9), were placed between theGBSS promoter and UbiT. The resulting expression cassette for the leadersequence associated with R1 is shown in FIG. 3 (SEQ ID NO.: 10). Asimilar cassette containing a spacer derived from the GBSS promoter (SEQID NO.: 11)—instead of the Ubi intron—separating the sense and antisensecopies of the R1 trailer is shown in (FIG. 3; SEQ ID NOs.: 12).Additional variants with a longer version of the GBSS promoter (SEQ IDNO.: 13) are shown in FIG. 3 (SEQ ID NOs.: 14-15).

To test the efficacy of the R1-associated leader in limiting acrylamideproduction, the expression cassette shown in FIG. 3 was inserted asKpnI-XbaI fragment between T-DNA borders of a binary vector. AnAgrobacterium LBA4404 strain carrying the resulting vector pSIM332 wasused to transform Russet Ranger potato. To induce tuber formation, 25shoots representing independent transformation events were transferredto soil and placed in a growth chamber (11 hours light, 25° C.). Afterthree weeks, at least 3 minitubers/line were stored for 4 weeks at 4° C.to induce starch mobilization. The glucose levels in these cold-storedminitubers were subsequently determined as described in Example 4, andcompared with the average glucose levels in untransformed plants andvector controls. As shown in Table 8, minitubers derived from all 25lines displayed reduced levels of glucose after cold-storage. Anapproximate 2-fold reduction in acrylamide levels in expected in Frenchfries derived from minitubers displaying reduced R1 expression levelscompared to controls. Much stronger effects of down-regulating R1 geneexpression are anticipated in mature tubers.

As an alternative to the leader-based approach, expression cassettesthat contained both a sense and antisense copy of the trailer sequenceassociated with R1 were generated. This trailer was obtained byperforming a reverse transcription polymerase chain reaction (RT-PCR) ontotal RNA isolated from microtubers of the potato cultivar RussetRanger. Complementary DNA was generated using the Omniscript RT Kit(Qiagen, CA) and then used as a template for a PCR reaction with Hotstart DNA polymerase (Qiagen, CA) with the gene-specific reverse primerR1-1 (5′-GTTCAGACAAGACCACAGATGTGA-3′). Sequence analysis of theamplified DNA fragment, cloned in pGEM-T demonstrated that the trailerassociated with R1 consists of 333 basepairs (SEQ ID NO.: 16). The senseand antisense copies of the trailer were separated by either the Ubiintron or the GBSS spacer- and sandwiched between GBSS promoter and Ubi3terminator (FIG. 3; SEQ ID NOs.: 17-18). Similar versions with thelarger GBSS promoter are shown in FIG. 3 (SEQ ID NOs.: 19-20).

Glucose and acrylamide levels can be determined as described above.Tubers displaying about 50% or greater reductions in glucoseconcentrations are expected to also accumulate about 50% less acrylamideduring the frying process. The improved health and storagecharacteristics of modified plants can be confirmed in maturefield-grown tubers.

Phosphate levels in potato tubers can be determined by using AOAC Method995.11 Phosphorus (Total) in Foods (45.1.33 Official Methods of Analysisof AOAC International, 17th Edition). Samples are prepared by dry ashingin a muffle furnace followed with an acid digestion. The dissolvedsamples are then neutralized and treated with a molybdate-ascorbic acidsolution and compared to a series of phosphorus standards (treatedsimilarly). A dual beam spectrophotometer would be used for thecalorimetric analysis at 823 nanometers. A significant decrease inphosphate content, which is beneficial for the environment, is expected.

Example 6 Leader Sequence Associated with the L-Alpha GlucanPhosphorylase Gene

Using conventional transformation methods, this Example demonstratesthat a novel leader sequence associated with the potato L-alpha glucanphosphorylase gene can be used to effectively enhance the processing andhealth characteristics of potato tubers.

Previously, it was shown that cold-induced sweetening can be reducedthrough antisense expression of 0.9-kb fragments derived from alphaglucan phosphorylase genes (Kawchuk et al., U.S. Pat. No. 5,998,701,1999). However, the antisense expression of these relatively large DNAfragments is undesirable because they contain new and uncharacterizedopen reading frames that may impact the nutritional quality of foods ifexpressed in transgenic plants (Table 1).

As a safer approach to the one described above, small leader and trailersequences that are associated with a L-type glucan phosphorylase genewere isolated from RNA of mature tubers. The primer pair used for thispurpose is: 5′-GGATCCGAGTGTGGGTAAGTAATTAAG-3′ (SEQ ID NO. 72), and5′-GAATTCTGTGCTCTCTATGCAAATCTAGC-3′ (SEQ ID NO. 73). The resultantleader sequence of 273 bp was amplified and is shown in SEQ ID NO.: 21.Similarly, the “direct” primer, 5′-GGAACATTGAAGCTGTGG-3′ (SEQ ID NO.74), was used with an oligo-dT primer to amplify a 158 bp “trailersequence” that is associated with the L-type phosphorylase gene (SEQ IDNO.: 22).

Expression cassettes were then designed using these trailer or leadersequences to modify the expression of L-type phosphorylase gene and, inso doing, lowering acrylamide levels in fried products by limitingstarch mobilization. These cassettes were constructed in a similar wayas described in Example 5, and are depicted in FIG. 3 (SEQ ID Nos.:23-26). An Agrobacterium strain containing a binary vector with thisexpression cassette, designated pSIM216, was used to infect potatostems, and generate 25 transgenic plants. Minitubers derived from theseplants were stored for 4 weeks at 4° C. to induce cold-sweetening. Thecold-stored minitubers were then analyzed for glucose levels. As shownin Table 9, minitubers from all transgenic lines displayed reducedglucose levels.

Four lines that displayed at least 50% reduced glucose concentrations(lines 216-2, 216-5, 216-10, and 216-21) were used to assessprocessing-induced acrylamide levels. Although acrylamide levels infried tubers derived from the first three lines were similar to those ofcontrols, French fries that were derived from line 216-21 accumulatedonly 45% of the wild-type acrylamide levels (136 vs. 305 parts perbillion). These results confirm the experiments described in Example 4for tubers overexpressing the potato invertase inhibitor gene, in thatrelatively large reductions in glucose (and fructose) concentrations areneeded to limit the heating-induced acrylamide accumulation incold-stored minitubers. Because silencing of the phosphorylase gene isexpected to be more effective in mature “216” tubers, reductions inacrylamide levels are also anticipated to be more pronounced in theFrench fries produced from such tubers. The improved health and storagecharacteristics of modified plants can be confirmed in mature tubers.

Example 7 Modified Polyphenol Oxidase Gene

Using conventional transformation methods, this Example demonstratesthat a modified polyphenol oxidase gene lacking a functionalcopper-binding site can be used effectively to reduce bruisesusceptibility in tubers.

Previously, it was shown that black spot bruise susceptibility can bereduced through antisense expression of the 1.8-kb PPO gene (Steffens,U.S. Pat. No. 6,160,204, 2000). However, expression of the reversecomplement of this large gene is undesirable because it contains new anduncharacterized open reading frames encoding peptides consisting of morethan 100 amino acids, which may potentially impact the nutritionalquality of foods (Table 1). As a safer approach to the one describedabove, the PPO gene was modified to encode a non-functional protein.

The wild-type potato PPO gene was isolated from Russet Ranger by using apolymerase chain reaction (PCR) method. First, genomic DNA was isolatedfrom sprouts of Russet Ranger. The potato PPO gene was then amplifiedfrom the potato genomic DNA using DNA polymerase and oligonucleotideprimers: 5′: CGAATTCATGGCAAGCTTGTGCAATAG-3′ (PPO-F) (SEQ ID NO. 75), and5′-CGAATTCTTAACAATCTGCAAGACTGATCG-3′ (PPO-R) (SEQ ID NO. 76). These weredesigned to complement the 5′- and 3′-ends of the potato PPO gene. Theamplified 1.6 kb fragment was cloned into a pGEM-T EASY vector (Promega)and confirmed to represent a functional PPO gene by sequence analysis(SEQ ID NO.: 27).

The copper binding domain in potato PPO was identified by aligning thisprotein with a sweet potato PPO protein that was shown to containconserved Cysteine (Cys) residue at position 92, Glutamine residue (Glu)at position 236, and Histidine (His) residues at positions 88, 109, 118,240, 244 and 274 coordinating the two active site coppers (Klabunde etal., Nature Structural Biol., 5: 1084-1090, 1998). These Cys, Glu, andHis residues are also present in potato PPO.

The inactive PPO gene was created by using a PCR mutation replacementapproach. Three fragments were amplified by Proof Start Taq DNAPolymerase (Qiagen) using 3 pairs of primers and wild-type Russet RangerPPO as a template. The sequences of the first pair, designated P1-F andP2-R, respectively, are: 5′-GAGAGATCTTGATAAGACACAACC-3′ (SEQ ID NO. 77),and 5′-CATTACC¹ATAAGCC²CAC³TGTATATTAGCTTGTTGC-3′ (SEQ ID NO. 78) (1: “A”to “C” mutation, resulting in Cysteine to Glycine substitution atposition 186; 2: “A” to “C” mutation, resulting in Cysteine toTryptophan substitution at position 183; 3: “A” to “C” mutation,resulting in Histine to Glutamine substitution at position 182). Thesequences of the second pair, designated P3-F and P4-R, respectively,are 5′-GTGCTTATAGAATTGGTGGC-3′ (SEQ ID NO. 79), and5′-TAGTTCCCGGGAGTTCAGTG-3′ (SEQ ID NO. 80). The sequences of the thirdpair, designated P5-F and P6-R, respectively, are5′-CTCCCGGGAACTATAGG⁴AAACATTCCTCT⁵CGGTCCTGTCCACATCTGGTC-3′ (SEQ ID NO.81) and 5′-GTGTGATATCTGTTCTTTTCC-3′ (SEQ ID NO. 82) (4: “A” to “G”mutation, resulting in Glutamine to Glycine substitution at position326; 5: “A” to “T” mutation, resulting in Histine to Leucinesubstitution at position 330).

An 80 bp fragment was amplified using primer P1-F and P2-R and digestedwith BglII. This fragment contains one sticky end (BglII) and one bluntend, and carries three mutations in copper binding site I. A 0.4 kbfragment amplified using primer P3-F and P4-R and digested with XmaIcontains one blunt end and one sticky end (XmaI). A 0.2 Kb fragment wasamplified using primer P5-F and P6-R and digested with XmaI and EcoRV.This third fragment with a sticky end (XmaI) and a blunt end (EcoRV) hastwo mutations in copper binding site II. The BglII and EcoRV fragmentfrom cloned wild-type potato PPO was then replaced with the above threeligated PCR amplified fragments. The presence of a total of 5 pointmutations in the modified PPO gene was confirmed by sequence analysis(SEQ ID NO.: 28). To create an expression cassette for modified PPO(mPPO), the following four fragments were simultaneously ligatedtogether: (1) a BamHI-HindIII fragment containing the GBSS promoter, (2)a HindIII-SacI fragment containing mutant PPO, (3) a SacI-KpnI fragmentcontaining the Ubi-3 terminator, and (4) plasmid pBluescript, digestedwith KpnI and BamHI. This expression cassette was then inserted betweenborders of a binary vector to create pSIM314.

The efficacy of the mPPO gene expression cassette was assessed bytransforming Russet Ranger stem explants with pSIM314. Nodal cuttings oftransgenic plants containing this expression cassette were placed on MSmedium supplemented with 7% sucrose. After a 5-week incubation period inthe dark at 18° C., microtubers were isolated and assayed for PPOactivity. For this purpose, 1 g of potato tubers was pulverized inliquid nitrogen. This powder was then added to 5 ml of 50 mM MOPS(3-(N-morpholino) propane-sulfonic acid) buffer (pH 6.5) containing 50mM catechol, and incubated at room temperature with rotation for about 1hour. The solid fraction was then precipitated, and the supernatanttransferred to another tube to determine PPO activity by measuring thechange of OD-410 over time. As shown in Table 10, microtubers isolatedfrom some of the transgenic lines displayed a significantly reducedpolyphenol oxidase activity compared to either untransformed controls orcontrols transformed with a construct not containing the mutant PPOgene. The strongest reduction in PPO activity was observed in lines“314-9”, “314-17”, and “314-29”. To test whether expression of themutant PPO gene also reduced PPO activity in minitubers, rootedplantlets of transgenic lines were planted in soil and incubated in agrowth chamber for 4 weeks. A PPO assay on isolated minitubersdemonstrated that reduced PPO activity in microtubers correlated in mostcases with reduced activities in minitubers (Table 10). Transgenic linesdisplaying a reduced PPO activity can be propagated and tested both inthe greenhouse and the field to confirm the “low bruise” phenotype inmature tubers. Because micro- and minitubers express a variety ofpolyphenol oxidases, some of which share only limited sequence homologywith the targeted polyphenol oxidase that is predominantly expressed inmature tubers, an even more profound reduction of PPO activity may beanticipated in the mature tubers of lines such as “314-9” and “314-17”.The data indicate that overexpression of a functionally inactive PPOgene can result in reduced bruise susceptibility. The improved healthand storage characteristics of modified plants can also be confirmed inmature field-grown tubers.

Example 8 Trailer Sequence of a Polyphenol Oxidase Gene that is Specificfor the Non-Epidermal Tissues of Potato Tubers

Using conventional transformation methods, this Example demonstratesthat a novel trailer sequence associated with the potato PPO gene can beused effectively to reduce bruise susceptibility in tubers.

Reverse transcription PCR was used to also isolate the trailer sequenceassociated with the PPO gene expressed in potato tubers. The primers forthe first PCR reaction were PPO-1 (5′-GAATGAGCTTGACAAGGCGGAG-3′, (SEQ IDNO. 83)) and oligo-dT; primers for a second nested PCR reaction werePPO-2 (5′-CTGGCGATAACGGAACTGTTG-3′, (SEQ ID NO. 84)) and oligo-dT.Sequence analysis of the amplified DNA fragments cloned into pGEM-Trevealed the presence of a 154-bp trailer (SEQ ID NO.: 29). A sense andantisense copy of this trailer, separated by the Ubi intron, was thenfused to the GBSS promoter and Ubi3 terminator as described above togenerate an expression cassette shown in FIG. 3 (SEQ ID NO.: 30). Analternative construct containing the trailer segments separated by aGBSS spacer is shown in FIG. 3 (SEQ ID NO.: 31). Similar versions withthe larger GBSS promoter are shown in FIG. 3 (SEQ ID NOs.: 32-33).Interestingly, the trailer of the PPO gene that is predominantlyexpressed in mature tubers (indicated with P-PPO3 in FIG. 4) isdifferent from the trailer of PPO genes that are predominantly expressedin other tissues including microtubers (indicated with PPOM-41 andPPOM-44 in FIG. 4). Because of the low homology between trailersassociated with different PPO genes, the use of the P-PPO3 trailer willresult in a silencing of the mature tuber-specific PPO gene only. Thisvery specific gene silencing would be difficult to accomplish withsequences derived from the PPO gene itself, thus demonstrating theadvantage of using non-coding sequences for gene silencing. To visualizethe extend of PPO activity, 0.5 mL of 50 mM catechol was pipetted on thecut surfaces of sliced genetically modified minitubers. Compared tocontrols, visual browning of the tuber regions was about 5 to 10-foldreduced. Interestingly, though, no reduced browning was observed in thepotato skin. It appears that the trailer sequence used specificallysilenced the PPO gene that is predominantly expressed in cortex and pithbut not in the epidermal skin. This unexpected finding may be beneficialfor tubers to protect themselves against some pathogens attempting toinfect through the skin because the PPO gene may play some role incertain defense responses. To quantitatively determine PPO activity, anassay was performed as described in Example 7. Table 11 shows up to 80%reduction of PPO activity in transformed minitubers compared tountransformed controls. The level of reduction is expected to be evengreater in mature tubers because these tubers express the targeted PPOgene more predominantly than mini- and microtubers. The improvedcharacteristics of lines such as “217-7” and “217-26” can be confirmedin mature tubers.

Example 9 An Expression Cassette to Increase Levels of Resistant Starch

Increasing the amylose/amylopectin ratios in tubers can further enhancethe nutritional value of potato products. One method that makes itpossible to increase amylose content is based on the antisenseexpression of genes encoding for the starch branching enzyme (SBE) I andII (Schwall et al., Nature Biotechnology 18: 551-554, 2000). Thedisadvantages of this method are that (1) the efficiency ofsimultaneously silencing two different genes through exploitation ofantisense technologies is very low, (2) the antisense expression of therelatively large SBE-I and SBE-II gene sequences results in theundesirable expression of open reading frames (Table 1) (3)corresponding constructs that harbor the two antisense expressioncassettes are unnecessarily large and complex, thus, increasing chancesof recombination and lowering transformation frequencies.

Our approach to increase amylose content in potato is based on theexpression of the trailer sequences that are associated with both genes.These trailers (SEQ ID No.:34 and 35) were isolated with the primerpairs 5′-GTCCATGATGTCTTCAGGGTGGTA-3′ (SEQ ID NO. 85), and5′-CTAATATTTGATATATGTGATTGT-3′ (SEQ ID NO. 86), and5′-ACGAACTTGTGATCGCGTTGAAAG-3′ (SEQ ID NO. 87), and5′-ACTAAGCAAAACCTGCTGAAGCCC-3′ (SEQ ID NO. 88). A single promoter drivesexpression of a sense and antisense fusion of both trailers, separatedby the Ubiquitin-7 intron, and followed by the Ubiquitin-3 terminator.The size of the entire expression cassette is only 2.5-kb.

Example 10 Development of Marker-Free Transformation Methods

This Example demonstrates that plants can be transformed effectivelywithout to need for stable integration of selectable marker genes.

This method is the first to take advantage of the phenomenon that DNAstargeted to the nuclei of plant cells often fails to subsequentlyintegrate into the plant cell's genome. The inventors made thesurprising discovery that it is possible to select for cells thattemporarily express a non-integrating T-DNA containing a selectablemarker gene by placing infected explants for 5 days on a plant mediumwith the appropriate selective agent. A second phenomenon that wasapplied to develop the current method is that T-DNAs from differentbinary vectors often target the same plant cell nucleus. By using twodifferent binary vectors, one containing the selectable marker on aT-DNA, and the other one carrying a T-DNA or P-DNA with the actualsequences of interest, it was possible to apply a transient selectionsystem and obtain populations of calli, shoots or plants, a significantportion of which represents marker-free transformation events.

A conventional binary vector designated pSIM011 was used to representthe vector with the “sequence of interest”, which is, in this test case,an expression cassette for the beta glucuronidase (GUS) gene located ona conventional T-DNA. The second binary vector that was used for theseexperiments contains an expression cassette comprising the neomycinphosphotransferase (NPTII) gene driven by the strong promoter of theUbiquitin-7 gene and followed by the terminator sequences of thenopaline synthase (nos) gene between the borders of the T-DNA of apSIM011-derivative.

Surprisingly, a strong level of transient NPTII gene expression levelscould also be obtained by replacing the nos terminator with theterminator of the yeast alcohol dehydrogenase 1 (ADH1) gene (Genbankaccession number V01292, SEQ ID NO. 56). This finding is interestingbecause the yeast ADH1 terminator does not share homology with any plantterminator. Importantly, it should be noted here that many yeastterminators do not function adequately in plants. For instance, almostno GUS gene expression was observed in a similar experiment as describedabove with the GUS gene followed by the yeast iso-1-cytochrome c (CYC1)terminator (Genbank accession number SCCYT1). An improved vectorcarrying the selectable marker gene NPTII was generated by replacing thenos terminator with the yeast ADH1 terminator. The binary vectorcontaining a selectable marker gene for transient transformation isdesignated “LifeSupport” (FIG. 5).

Potato stem explants were simultaneously infected with two A.tumefaciens LBA4404 strains containing pSIM011 and LifeSupport,respectively. A 1/10 dilution of overnight-grown cultures of each strainwere grown for 5-6 hours before they were precipitated, washed andresuspended an OD_(600nm) of 0.4 as described in Example 3. Theresuspended cells were then used to infect 0.4-0.6 cm internodal potatosegments at a final density of each bacteria of 0.2 (OD_(600nm)).Infected stems were treated as in Example 3 with a main difference: theselection with kanamycin was limited to the first 5 days of culture oncallus induction medium. Then, explants were allowed to further developin fresh CIM and SIM containing only timentine 150 mg/L but no selectiveantibiotic. Within about 3 months from the infection day leaves fromshoots derived from calli developed in 40-60% of the infected stems wereboth tested for GUS expression and PCR analyzed to identify events thatcontained the sequences of interest but no marker gene. As shown inTable 12, 11% of shoots represented marker-free transformation events.

The two-strain approach described above was also used to transformtobacco. Shoots that developed within about 2 months were GUS assayedand PCR analyzed. The high frequency of marker-free transformationevents identified (18%) implies that the developed method is applicableto plant species other than potato (Table 12).

Importantly, sequential rather than simultaneous infection with the twodifferent Agrobacterium strains resulted in an increase in theefficiency of marker-free transformation. The surprising effect ofsequential infections was discovered by infecting potato stem explantswith the Agrobacterium strain containing pSIM011, placing the infectedexplants on co-cultivation plates for 4 hours, and then re-infectingthem with the LifeSupport vector. The doubly infected explants weretreated as previously described in this example. As shown in Table 13,the lag time of 4 hours between the two different infections resulted ina 2-fold increased frequency of marker-free transformation events inpotato.

Example 11 Precise Breeding with pSIM340

This Example demonstrates the efficacy of precise breeding. The healthand agronomic characteristics of potato plants are enhanced by insertingpotato genetic elements (see Examples 1, 4, and 7) into potato, usingmarker-free transformation (Example 10).

A binary vector containing two expression cassettes for the invertaseinhibitor and mutant polyphenol oxidase genes inserted between P-DNAtermini, designated pSIM340 (FIG. 1), was created by inserting bothexpression cassettes of mutant PPO and invertase inhibitor into a binaryvector pSIM112′. Potato stem explants were infected simultaneously withpSIM340 and a further improved LifeSupport vector. The infected explantswere then co-cultivated, subjected to transient selection, and inducedto proliferate and develop shoots as discussed earlier. After 3 months,small shoots were transferred to new media and allowed to grow for 3additional weeks. Shoots were then phenotypically analyzed, and leafmaterial was collected for molecular analyses to determine the presenceof backbone, marker gene and P-DNA with the sequences of interest, asdescribed in Examples 2 and 3. As shown in Table 14, 1.2% of eventsrepresented a plant that contained the modified P-DNA of pSIM340 withoutLifeSupport. This frequency of maker-free transformation is lower thanfound for a T-DNA, again revealing a functional difference between P-DNAand T-DNA.

Example 12 Selecting Against Stable Integration of LifeSupport T-DNAs

This Example demonstrates that the efficiency of precise breedingmethods can be increased by selecting against stable integration ofLifeSupport T-DNAs using the bacterial cytosine deaminase gene.

The previous example demonstrates that the efficiency of marker-freetransformation is several-fold lower with a modified P-DNA than with aconventional T-DNA. To improve the efficiency of generating shoots onlycontaining a modified P-DNA, an expression cassette for a silicide genefusion comprising the bacterial cytosine deaminase (codA) and uracilphosphoribosyltransferase (upp) genes (InvivoGen, CA) was insertedbetween T-DNA borders of the LifeSupport vector, generating pSIM346(FIG. 5). Potato stem explants were infected with one strain carryingpSIM340 and the other carrying pSIM346, and subsequently placed on thefollowing media: (1) co-cultivation media for 2 days, (2) CIMTK media toselect for. transient marker gene expression for 5 days, (3) CIMT mediato allow proliferation of plant cells that transiently expressed themarker gene for 30 days, (4) SIMT media with 500 mg/L of non-toxic5-fluorocytosine (5-FC), which will be converted by plant cellsexpressing codA::upp into the toxic toxic 5-fluorouracil (5-FU), toselect against stable integration of the LifeSupport TDNA. Callus gaverise to shoots on SIMT within 4 weeks. These shoots were transferred toMS media with timentin and allowed to grow until sufficient tissue wasavailable for PCR analysis. DNA was then extracted from 100 shoots andused to determine the presence of P-DNA, LifeSupport and backbone. Asshown in Table 15, none of the shoots analyzed contained a LifeSupportT-DNA, indicating, for the first time, that the codA::upp gene fusioncan be used as negative selectable marker prior to regeneration. Moreimportantly, our results demonstrate that a negative selection againstLifeSupport T-DNA integration increases the frequency of shoots thatonly contain a modified P-DNA. By coupling a positive selection fortransient marker gene expression with a negative selection againststable integration of the codA::upp gene fusion, the frequency of shootsonly containing a modified P-DNA is about 5-fold higher than by onlyemploying the positive selection for transient marker gene expression(Table 15).

An even greater increase in the efficiency of marker-free transformationwas obtained by using the LifeSupport vector pSIM350 (FIG. 5), which issimilar to pSIM346 but contains the codA gene instead of the codA::uppgene fusion. Potato stem explants simultaneously infected with pSIM340and pSIM350 were treated as described above, and 51 resulting shootswere molecularly tested for the occurrence of events only containing theT-DNA region from pSIM340. Interestingly, this PCR analysis revealedthat some shoots contained the codA gene (Table 15). This findingdemonstrates that codA is not as tight a negative selectable marker ascodA::upp in plants. More importantly, a large number of shoots (29%)were shown to represent marker-free transformation events.

Efficiencies can be further increased by not infecting explantssimultaneously with pSIM340 and pSIM350 but sequentially. By infectingthe explants with pSIM340 and re-infecting them with pSIM350 after 4hours, marker-free transformation frequencies are expected to beapproximately 30-40%.

Example 13 Impairing Integration of LifeSupport T-DNAs

This Example demonstrates that the efficiency of precise breedingmethods can be increased by impairing integration of the LifeSupportT-DNA into the plant genome using an omega-mutated virD2 gene.

It has been shown that the omega domain of the Agrobacterium proteinvird2 is important for the ability of that protein to support T-DNAintegration into plant genomes (Mysore et al., Mol Plant MicrobeInteract 11: 668-83, 1998). Based on this observation, modifiedLifeSupport vectors were created that contain an expression cassette foran omega-mutated virD2 protein inserted into the SacII site in theirbackbone sequences. The expression cassette was obtained by amplifying a2.2-kb DNA fragment from plasmid pCS45 (courtesy of Dr. Walt Ream—OregonState University, OR, USA—, SEQ ID NO.: 36). A LifeSupport-derivativecarrying this expression cassette, designated pSIM401Ω (FIG. 5), wasused to support the transformation of potato plants with the modifiedP-DNA of pSIM340. After transient selection and shoot induction, 100shoots were molecularly tested for the presence of transgenes. As shownin Table 15, 4.4% of shoots only contained the modified P-DNA,indicating that the use of omega-virD2 increases the efficiency ofmarker-free transformation about 4-fold (Table 15).

Efficiencies are further improved by increasing the size of theLifeSupport T-DNA from 3.7 kb (in pSIM401Ω) to 8.1 kb (in thepSIM401Ω-derivative designated pSIM341Ω; FIG. 5). By regenerating shootsfrom potato stem explants simultaneously infected with pSIM340 andpSIM341Ω, 7 of 81 analyzed events (7%) were shown to representmarker-free transformation events (Table 15).

A further improvement can be obtained by infecting explants sequentiallyrather than simultaneously with pSIM340 and LifeSupport. In a similarway as described in Example 10, the frequency of plants that onlycontain a modified P-DNA can be about doubled by infecting the explantswith pSIM340 and re-infecting them with LifeSupport after 4 hours.

Example 14 Development of a 1-Strain Approach

This Example demonstrates that high frequencies of marker-freetransformation can also be obtained by using a single Agrobacteriumstrain that contains both the P-DNA vector and LifeSupport

Two compatible binary vectors were created that can be maintainedsimultaneously in Agrobacterium. Instead of using this system to stablyintegrate two T-DNAs carrying the DNA-of-interest and a marker gene,respectively (Komari et al. U.S. Pat. No. 5,731,179, 1998), it isintended for integration of only the modified P-DNA.

The first vector, designated pSIM356, contains an expression cassettecomprising the GUS gene driven by the Ubi7 promoter and followed by UbiTinserted between P-DNA termini. The backbone portion of this vectorcontains bacterial origins of replication from pVS1 and pBR322, aspectinomycin resistance gene for bacterial selection, and an expressioncassette for the IPT gene to enable selection against backboneintegration in plants (FIG. 1). The second vector, designated pSIM363,contains an expression cassette comprising the NPTII gene driven by theUbi7 promoter and followed by the yeast ADH1 terminator inserted betweenconventional T-DNA borders (FIG. 5). The backbone portion of this vectorcontains bacterial origins of replication from ColE1 (Genbank numberV00268) and ori V (Genbank number M20134), and a kanamycin resistancegene for bacterial selection.

The concept of increasing marker-free transformation frequencies usingpSIM356 and pSIM363 was tested in 100 tobacco shoots. As shown in Table16, about 19% of regenerated shoots contained the DNA of interestwithout marker gene. An increase in marker-free transformationefficiency was also found by applying this 1-strain approach to potato.Nine of 60 independent shoots tested (15%) contained the pSIM340 T-DNAand lacked the LifeSupport T-DNA (Table 16).

The 1-strain approach can be combined with the method described inExample 12 to couple a positive selection for transient marker geneexpression with a negative selection against stable integration of thecodA gene. For this purpose, the LifeSupport vector pSIM365 wasdeveloped (FIG. 5). An Agrobacterium strain carrying this vectortogether with a P-DNA vector can be used to efficiently develop plantsthat only contain an expression cassette-of-interest located within aP-DNA stably integrated in their genomes.

Example 15 Precise Breeding Method Relying on a Native Marker

Apart from transforming crop plants with P-DNAs that only contain thedesirable sequences to introduce beneficial traits, the presentinvention also provides a method of transforming such plants with P-DNAsthat contain an additional native marker gene. Our novel and nativemarker genes of choice are potato homologs of the Arabidopsis vacuolarNa+/H+ antiporter gene and alfalfa alfin-1 gene. Expression of thesegenes do not only allow the identification of transformation events, butalso provides salt tolerance to transformed plants. High salinity levelsin an increasing acreage of agricultural land will therefore less affectpotato plants containing the salt tolerance marker.

Two versions of a vacuolar Na+/H+ antiporter homolog, designated Pst(Potato salt tolerance) were amplified from cDNA of a late blightresistant variety obtained from the US Potato Genbank (WI), designated“LBR4”, using the oligonucleotide pair 5′-CCCGGGATGGCTTCTGTGCTGGCT-3′(SEQ ID NO. 89) and 5′-GGTACCTCATGGACCCTGTTCCGT-3′ (SEQ ID NO. 90).Their sequences are shown in SEQ ID NO.:37 and 38. A third gene (SEQ IDNO.:39) with homology to alfin-1 was amplified from LBR4 potato DNAusing the primers 5′-CCCGGGTATGGAAAATTCGGTACCCAGGACTG-3′ (SEQ ID NO. 91)and 5′-ACTAGTTAAACTCTAGCTCTCTTGC-3′ (SEQ ID NO. 92). The efficacy of thePst genes to function as transformation marker was assessed by insertinga fusion with the Ubi7 promoter between conventional T-DNA borders of amodified pSIM341 vector. After a transient selection period,kanamycin-resistant cells are allowed to proliferate and develop shoots.These shoots are then transferred to media that contain 100-150 mMsodium chloride. Salt-tolerant shoots represent transformation eventsthat contain the T-DNA of the modified pSIM341.

Example 16 Tuber-Specific Promoter

A newly isolated tiber-specific promoter can replace the GBSS promoterused to develop the expression cassettes described in previous examples.This promoter was isolated from the genome of Russet Burbank potatoplants by using the inverse polymerase chain reaction with primersspecific for a potato proteinase inhibitor gene (Genbank AccessionD17332) (SEQ ID NO. 39). The efficacy of the PIP promoter was tested bycreating a binary vector that contains the GUS gene driven by thispromoter and an expression cassette for the NPTII marker gene. A similarconstruct with the PIP promoter replaced by the GBSS promoter was usedas control. Transformed shoots were obtained by infecting stem explantswith Agrobacterium strains carrying the binary vectors, co-cultivationfor 2 days, and selection on CIMTK medium for 2 months. These shootswere transferred to new media to induce root formation, and then plantedinto soil. Tubers can be assayed for GUS expression after a 3-monthgrowth period in the green house.

Example 17 Preferred Constructs and Transformation Methods for PreciseBreeding

Apart from pSIM340, many other vectors can be used to improve potatoplants by transforming them with modified P-DNAs. Two of such vectorscontain an expression cassette for a sense and antisense copy of thetrailer associated with a PPO gene that is expressed in all tubertissues except for the epidermis (see Example 8). Vector pSIM370contains an additional expression cassette for a sense and antisensecopy of the leader associated with phosphorylase gene (see Example 6).Vector pSIM371 contains a third expression cassette for the potatoalfin-1 homolog (FIG. 1).

A third alternative vector, designated pSIM372, contains both anexpression cassette for the potato alfin-1 homolog, and an expressioncassette for a sense and antisense copy of a fusion of thePPO-associated trailer, R1-associated leader, andphosphorylase-associated leader.

The preferred LifeSupport vector for a 1-strain approach is pSIM365. Fora 2-strain approach, the preferred vector is pSIM367, which containsexpression cassettes for both NPTII and codA between T-DNA borders, andan additional expression cassette for omega virD2 in the plasmidbackbone (FIG. 5).

Potato stem explants are infected with 1 strain carrying both pSIM365and any of the vectors pSIM370, 371, and 372, or sequentially with 2strains carrying pSIM366 and any of the preferred vectors-of-interest,respectively. After a 2-day co-cultivation and a 5-day transientselection period, the explants are transferred to media forproliferation/regeneration and elimination of Agrobacterium. Thirty dayslater, explants are transferred again to the same media but now alsocontaining 5-FU to eliminate events containing LifeSupport T-DNAs.Shoots that subsequently arise on calli are transferred to regenerationmedia that may contain 100-200 mM salt to screen for salt tolerantevents. The IPT-negative shoots are allowed to root and develop intomature plants. A large proportion of these plants (10%-100%) arepredicted to represent marker-free and backbone-free plants containing aP-DNA with nucleotide sequences of interest stably integrated into theirgenomes. TABLE 1 Potentially expressed uncharacterized peptides inantisense potato lines Gene (size of Predicted peptides encoded byfragment used) ORFs in reverse-complemented DNA R1 (1.9-kb) MSSTSNVGQDCLAEVTISYQ WVGRVINYNF FLLIHWYTVV EASTGITFQI FPIGIRSEDD RSFYEKADRF AWVTMSSESTFSKT PNGRATDVGI PTEEGTFPFR YAILRDLAPT ISLVNSSADI A MSEGVGFKSKILPSFAWRSA NILGSKHVAK QTFPFLARTE TCERTSGMSG VIRATAPSGI SSSPLTDFAT KIVGFSGLTP (1-kb) VCSPALKADK SKSADGTCVD HSRRLIVVLV LYPGMGTSYA TAFISSPPIQYLFPSDPVET FP MLGSLVLPKS PENRKQAVPN PHFQEQHLVP EKPHFLDCGQ GFSKLPQMHQMVNFLTQGIV DMETAFGSPK MGGFGKEQFG ACVSRSEMDE SGIGAVMVEQ VCSICSRHFV LSMQIGHTP (0.9-kb) MLEGSMWPWN QESMKRAFLN HHFLMLHLFP AQRPPQAADP VCLKHQHMHCGCLSFQLHLS KLAPGDTPLI SSMFALD MKLCSSIILS IIKQKQVEIL RACFGFPETKTISVFSSVSW NWHIICKSL MTKKPDRKDN IMPYNFPGTK FLQPIFRNFF LPSLCDKLLKKSISVPQAIT PCWKVQCGHG IKKA PPO (1.8-kb) TILKLDLHTF NGHFFTASFW NQSHRNSIFIFQSHILQQFS YRQLESNTGN MISITSMNM RQASITPCKL RLIKLICIHS LVHVQKHIEPYIVPIIIRYF IECQYLLLLI FLLCCP MKGKEKPREM NLQFFTTNFV STVAISTMNI SLLFKAKRVKGVFIKFPHST RSQLILGYVL LIRRMSRGAD AEFSHRRELV VRNTIDLIGY RRATTVYYINTFFYMGSTTR LEIRRWYRCS SR MEWALARNRI PFFYCPNSLR TSHGKGYDFH RRKRIQSSTNLYLLNPFFSR QLISIHSTSC PHWHGGSKKS DLNRVSRNYP CLHRFFDEVC HRSRCEPEYE GCFQSBE A (1.2-kb) MNNITHSPIL IPFLEQLNPF ISNCHMQPIV KANTPILNGN TKCRHSANIFTNGNCIWEKP MNKIVDQHQI HNSIHISCES KVFLVVPSES HR MKFRYPSPPN PIVTSLIILCNAIPRSINDV DGLSRAIKSY ISLSISQNAI VLSPTRA SBE B (2.6-kb) MVNIMTSSSMATKFPSITVQ CNSVLPWQVT SNFIPFVCVL WVEVEYKYQV TTFKHNNLII IIHAAYYLFSMAKLVTHEIE VPLSSQGHCE KMDHLVKRNS SINNRRSICQ ARHARIHLFV H MFETKLNSGVVWNDWLTVNI RNSNTPNTKL VLLHHVVRTV PSIEIANNFV FLSSRSPFTI DYATIFPVES KFMLYTSLYISY LSNSMLLPSW TNLHHSYSLN NLSTYLGLPL PGGNQNQFLP QKQGCQGPAY QKHLRQ

TABLE 3 Transformation efficiency Calli/tobacco leaf Calli/potato stemBinary vector explant ± SE explant ± SE pBI121  7.8 ± 0.6 0.31 ± 0.10pSIM108 10.2 ± 0.6 0.59 ± 0.07 pSIM109 12.8 ± 0.6 0.47 ± 0.05

TABLE 4 Backbone integration resulting from Russet Ranger transformationBinary Total PCR⁺ for 0.6 kb vector Nr. IPT phenotype PCR⁺ for IPTbackbone fragment pBI121 98 NA NA 54 (55%) pSIM108 193 138 (71%) 137(71%) NA pSIM109 133  82 (62%)  80 (60%) NANA: not applicable

TABLE 5 Backbone integration resulting from Russet Burbanktransformation Binary vector Total Nr. IPT phenotype PSIM108 79 49 (60%)PSIM109 72 60 (84%)

TABLE 6 Acrylamide levels in French fries derived from cold-storedpSIM320 minitubers Line glucose mg/g (%-reduced) acrylamide (PPB)Untransformed 10.2 469 Vector control 10.2 NA 320-2 5.4 (47%)  95 320-45.8 (43%) 107 320-7 8.7 (14%) 353 320-9 7.4 (27%) 137 320-17 6.0 (41%)506 320-21 8.5 (16%) 428 320-33 6.6 (35%) 516NA: not available

TABLE 7 Acrylamide levels in French fries derived from untransformedmature tubers Stored at 18° C. Stored at 4° C. (color id.*) (color id.*)Glucose levels <0.1 mg/g 3.4 mg/g 8-minute blanch 53 PPB (78) 603 PPB(56) 12-minute blanch 28 PPB (84) 244 PPB (71)*a higher value indicates a lighter color of the finished Fry product

TABLE 8 Glucose levels in cold-stored pSIM332 minitubers Line glucosemg/g (%-reduced) Untransformed control 11.6 ± 0.5 Vector control 11.5 ±0.5 332-1 5.4 (53%) 332-2 4.8 (58%) 332-4 7.0 (39%) 332-5 5.8 (50%)332-6 6.9 (40%) 332-7 6.0 (48%) 332-8 6.8 (41%) 332-9 6.6 (43%) 332-105.4 (53%) 332-11 6.1 (47%) 332-12 6.4 (44%) 332-13 6.4 (44%) 332-15 7.7(33%) 332-16 6.5 (43%) 332-17 5.3 (54%) 332-18 7.1 (38%) 332-21 6.3(46%) 332-22 5.4 (53%) 332-23 4.2 (63%) 332-31 6.0 (48%) 332-34 6.2(48%) 332-35 6.4 (44%) 332-39 6.7 (41%) 332-40 7.5 (35%) 332-41 5.7(50%)

TABLE 9 Glucose levels in cold-stored pSIM216 minitubers Line glucosemg/g (%-reduced) Untransformed control 11.6 ± 0.5 Vector control 11.5 ±0.5 216-2 5.5 (52%) 216-3 8.8 (23%) 216-4 7.4 (36%) 216-5 5.8 (50%)216-8 8.4 (27%) 216-10 5.1 (56%) 216-11 10.1 (19%)  216-12 9.3 (19%)216-13 6.4 (44%) 216-15 8.8 (23%) 216-16 9.7 (16%) 216-17 6.4 (44%)216-19 8.7 (24%) 216-21 3.2 (72%) 216-24 9.4 (18%) 216-26 9.3 (19%)216-29 7.1 (38%) 216-30 8.2 (29%) 216-32 9.3 (19%) 216-34 7.1 (38%)216-35 7.8 (32%) 216-38 7.1 (38%) 216-42 8.1 (30%) 216-44 9.4 (18%)216-45 10.2 (11%) 

TABLE 10 PPO activity in potato lines expressing a modified PPO geneOD-410/gram Line micro-tubers (%-reduced) mini-tubers (%-reduced)Untransformed 24.59 ± 2.22 20.07 ± 1.21 controls Vector controls 22.59 ±3.36 19.55 ± 1.43 314-1 2.36 (90%) 17.8 (11%) 314-2 41.52 (−76%) 21.3(−7%) 314-4 18.40 (22%) 5.4 (73%) 314-5 8.49 (64%) 19.1 (4%) 314-7 16.04(32%) 16 (20%) 314-8 14.86 (37%) 17 (15%) 314-9 5.43 (77%) 4.3 (78%)314-12 19.35 (18%) 19.6 (2%) 314-13 18.17 (23%) 15.4 (23%) 314-14 18.64(21%) 17.32 (13%) 314-16 13.92 (41%) 18.2 (9%) 314-17 5.19 (78%) 2.4(88%) 314-20 26.66 (−13%) 13.2 (34%) 314-21 11.32 (52%) 17.6 (12%)314-22 13.45 (43%) 18.8 (6%) 314-23 5.19 (78%) 20.4 (−2%) 314-24 15.10(36%) 19.6 (2%) 314-25 23.12 (2%) 19 (5%) 314-26 13.45 (43%) 17.8 (11%)314-27 26.42 (−12%) 19.4 (3%) 314-28 31.85 (−35%) 19.4 (3%) 314-29 3.77(84%) 14.8 (26%) 314-31 23.83 (−1%) 21.2 (−6%) 314-32 28.78 (−22%) 20(0%)

TABLE 11 Table 11. PPO activity in potato minitubers expressing amodified trailer sequence associated with the PPO gene Line OD-410/gram(%-reduced) Untransformed controls 20.6 ± 1.3 Vector controls 17.9 ± 2.1217-1 12.5 (39.4%) 217-4 12.6 (38.6%) 217-5 11.3 (45.0%) 217-6 6.1(70.4%) 217-7 5.7 (72.5%) 217-9 10.4 (49.6%) 217-10 15.2 (26.3%) 217-1115.2 (26.3%) 217-12 6.6 (67.9%) 217-14 15.4 (25.4%) 217-15 13.5 (34.6%)217-16 6.0 (71.0%) 217-17 9.7 (53.0%) 217-19 8.6 (58.4%) 217-21 14.2(31.1%) 217-22 9.7 (53.0%) 217-23 15.2 (26.3%) 217-24 8.2 (60.1%) 217-2511.9 (42.2%) 217-26 3.1 (84.8%) 217-27 6.2 (69.9%) 217-29 7.2 (65.1%)

TABLE 12 Marker-free transformation with the LifeSupport vector +pSIM011 Marker Gene-of-interest Plant Co-transformed only onlyUntransformed Potato  0% 33% 11% 56% Tobacco 20% 26% 18% 36%Co-transformed: PCR-positive for both GUS and NPTGene-of-interest only: PCR-positive for GUSUntransformed: Plants are PCR-negative for both GUS and NPT

TABLE 13 Sequential potato transformation with the LifeSupport vectorand pSIM011 Time Marker Gene-of-interest window Co-transformed only onlyUntransformed O hrs  9% 36%  9% 46% 4 hrs 20% 30% 20% 30%Untransformed: Plants are PCR-negative for marker and gene-of-interest

TABLE 14 Marker-free transformation with the P-DNA vector pSIM340 +LifeSupport Marker Gene-of-interest Plant Co-transformed only onlyUntransformed Potato 17% 52.8% 1.2% 29%Co-transformed: PCR-positive for both the PPO gene of pSIM340 and theNPT gene from LifeSupportUntransformed: Plants are PCR-negative for PPO and NPTII

TABLE 15 Marker-free potato transformation with pSIM340 + improvedLifeSupport vectors LifeSupport Co- Marker Gene-of-interest vectortransformed only only Untransformed PSIM346  0%  0% 4% 96% PSIM350 10%10% 29%  51% PSIM401Ω  6% 34% 5% 55% pSIM341Ω 16% 23% 7% 54%Co-transformed: PCR-positive for both the PPO gene of pSIM340 and theNPT gene from LifeSupportUntransformed: Plants are PCR-negative for PPO and NPTII

TABLE 16 Marker-free potato transformation with a single Agrobacteriumstrain carrying both pSIM356 and pSIM363 Marker Gene-of-interest PlantCo-transformed only only Untransformed Tobacco 50% 15% 19% 16% Potato22%  5% 15% 58%Co-transformed: PCR-positive for both the GUS gene of pSIM356 and theNPT gene from LifeSupportUntransformed: Plants are PCR-negative for PPO and NPTIISEQ ID NOs.

-   SEQ ID NO.: 1: Potato P-DNA. The bold underlined portions of SEQ ID    NO. 1 represent the left (5′-) and right (3′-) border-like sequences    of the P-DNA respectively.-   SEQ ID NO.: 2: Wheat P-DNA-   SEQ ID NO.: 3: Expression cassette for the IPT gene-   SEQ ID NO.: 4: Binary vectors pSIM111-   SEQ ID NO.: 5: Potato invertase inhibitor gene-   SEQ ID NO.: 6: Potato GBSS promoter-   SEQ ID NO.: 7: Potato Ubiquitin-3 gene terminator-   SEQ ID NO.: 8: Potato leader associated with the R1 gene-   SEQ ID NO.: 9: Potato Ubiquitin intron-   SEQ ID NO.: 10: Expression cassette for a sense and antisense copy    of the leader associated with the R1 gene-   SEQ ID NO.: 11: Spacer-   SEQ ID NO.: 12: Alternative expression cassette for a sense and    antisense copy of the leader associated with the R1 gene-   SEQ ID NO.: 13: Longer potato GBSS promoter-   SEQ ID NO.: 14: Alternative expression cassette for a sense and    antisense copy of the leader associated with the R1 gene-   SEQ ID NO.: 15: Alternative expression cassette for a sense and    antisense copy of the leader associated with the R1 gene-   SEQ ID NO.: 16: Potato trailer associated with the R1 gene-   SEQ ID NO.: 17: Expression cassette for a sense and antisense copy    of the trailer associated with the R1 gene-   SEQ ID NO.: 18: Expression cassette for a sense and antisense copy    of the trailer associated with the R1 gene-   SEQ ID NO.: 19: Expression cassette for a sense and antisense copy    of the trailer associated with the R1 gene-   SEQ ID NO.: 20: Expression cassette for a sense and antisense copy    of the trailer associated with the R1 gene-   SEQ ID NO.: 21: Potato leader associated with the L glucan    phosphorylase gene-   SEQ ID NO.: 22: Potato trailer associated with the L glucan    phosphorylase gene-   SEQ ID NO.: 23: Expression cassette for a sense and antisense copy    of the leader associated with the L glucan phosphorylase gene-   SEQ ID NO.: 24: Alternative expression cassette for a sense and    antisense copy of the leader associated with the L glucan    phosphorylase gene-   SEQ ID NO.: 25: Alternative expression cassette for a sense and    antisense copy of the leader associated with the L glucan    phosphorylase gene-   SEQ ID NO.: 26: Alternative expression cassette for a sense and    antisense copy of the leader associated with the L glucan    phosphorylase gene-   SEQ ID NO.: 27: Potato PPO gene-   SEQ ID NO.: 28: Modified inactive potato PPO gene-   SEQ ID NO.: 29: Potato trailer associated with a PPO gene-   SEQ ID NO.: 30: Expression cassette for a sense and antisense copy    of the trailer associated with a PPO gene-   SEQ ID NO.: 31: Alternative expression cassette for a sense and    antisense copy of the trailer associated with a PPO gene-   SEQ ID NO.: 32: Alternative expression cassette for a sense and    antisense copy of the trailer associated with a PPO gene-   SEQ ID NO.: 33: Alternative expression cassette for a sense and    antisense copy of the trailer associated with a PPO gene-   SEQ ID NO.: 34: Potato trailer associated with a starch branching    enzyme gene-   SEQ ID NO.: 35: Potato trailer associated with a starch branching    enzyme gene-   SEQ ID NO.: 36: Expression cassette for an omega-mutated virD2 gene-   SEQ ID NO.: 37: Potato salt tolerance gene Pst1-   SEQ ID NO.: 38: Potato salt tolerance gene Pst2-   SEQ ID NO.: 39: Potato salt tolerance gene Pst3-   SEQ ID NO.: 40: Potato tuber specific promoter-   SEQ ID NO.: 56: Yeast ADH terminator-   SEQ ID NO. 94: Wheat left border-like sequence

SEQ ID NO. 95: Wheat right border-like sequence SEQID1GTTTACAGTACCATATATCCTGTCA GAGGTATAGAGGCATGACTGGCATGATCACTAAATTGATGCCCACAGAGGAGACTTATAACCTACAGGGGCACGTACTTCTAGCACTTGAAAGTGACTGACCGTAGTCCAACTCGGTATAAAGCCTACTCCCAACTAAATATATGAAATTTATAGCATAACTGCAGATGAGCTCCATTCTAGAGTAGGTACCCAGCTCGAATTCCTTACTCCTCCACAAAGCCGTAACTGAAGCGACTTCTATTTTTCTCAACCTTCGGACCTGACGATCAAGAATCTCAATAGGTAGTTCTTCATAAGTCAGACTATCCTTCATAGCTACACTTTCTAAAGGTACGATAGATTTTGGATCAACCACACACACTTCG TTTACACC GGTATATATCCTGCCASEQID2 TGGCAGGATATATGAGTGTGTAAACAACCATAATCAGGCTGTAATTATCAAGAGAACTAATGACAAGAAGCAGAGCTTATCAAGTGTTTCGTCCAGCTGTAACATGGGCACAAAAGCTTGCTTGATGCATGTCTGGCTTTTCAAAGAGCAATGTATTCTCAGGTACCTGCACGTTTGATCCCCTACCACGTACAAGACGAGCAGAAACGACATGTCTGCAGAAACTTACACACATCCATTGCAGACTCGTTCCGAAGCATCAGGAGAGTAGTCAGCAATGGTCATCTGCTGATGTAAATTAATTGATTGTTGGTAATCAAATTTTAACAGCAATATATATAATATATCAATAGTATATTGAACTATGAAAGACTGTAATCATATATAACAGCATACAAATTGTCGTGGAAACAAGAGGAGCTCATCAAGTGTTTAGTTCAGAAATAGCTAACCAAGAATGCAATATAATAGGGGTACTGAGCTCCCTTCAAAATTACTAACTTCAGAAATAGCTAACCAAGAATGCAATGGCATTGCATAATTTAAACAACTGTCAGCACCAATCTCTGACTGAAGGCACTTTACCCATTCAGAAGAGCACACATTTTCTGAACGACAACTCTGAGCGGGGATTGTTGACAGCAGCAATTAATCTGGCCTCAAGATGGTTTCCAACAACATAGATCAGATACAGCACTCAAGCACCCAATAATCAGCCAGTACTGATCTGGTTACCACTGCAATTGATTAACAGATGAACTGTGAAATTAAGATTTAACTGACAGTAATATATACCAGTTGGCAGGATATATCCCTCTGTAAAC SEQID3CTGCAGCCAAAGCACATACTTATCGATTTAAATTTCATCGAAGAGATTAATATCGAATAATCATATACATACTTTAAATACATAACAAATTTTAAATACATATATCTGGTATATAATTAATTTTTTAAAGTCATGAAGTATGTATCAAATACACATATGGAAAAAATTAACTATTCATAATTAAAAAATAGAAAAGATACATCTAGTGAAATTAGGTGCATGTATCAAATACATTAGGAAAAGGGCATATATCTTGATCTAGATAATTAACGATTTTGATTTATGTATAATTTCCAAATGAAGGTTTATATCTACTTCAGAAATAACAATATACTTTTATCAGAACATTCAACAAAGTAACAACCAACTAGAGTGAAAAATACACATTGTTCTCTAAACATACAAAATTGAGAAAAGAATCTCAAAATTTAGAGAAACAAATCTGAATTTCTAGAAGAAAAAAATAATTATGCACTTTGCTATTGCTCGAAAAATAAATGAAAGAAATTAGACTTTTTTAAAAGATGTTAGACTAGATATACTCAAAAGCTATCAAAGCAGTAATATTCTTCTTACATTAAGTATTTTAGTTACAGTCCTGTAATTAAAGACACATTTTAGATTCTATCTAAACTTAAATGTATCTAGAATACATATATTTGAATGCATCATATACATGTATCCGACACACCAATTCTCATAAAAAGCGTAATATCCTAAACTAATTTATCCTTCAAGTCAACTTAAGCCCAATATACATTTTCATCTCTAAAGGCCCAAGTGGCACAAAATGTCAGGCCCAATTACGAAGAAAAGGGCTTGTAAAACCCTAATAAAGTGGCACTGGCAGAGCTTACACTCTCATTCCATCAACAAAGAAACCCTAAAAGCCGCAGCGCCACTGATTTCTCTCCTCCAGCCGAAGATGCAGATCTTCGTGAAGACCCTAACGGGGAAGACGATCACCCTAGAGGTTGAGTCTTCCGACACCATCGACAATGTCAAAGCCAAGATCCAGGACAAGGAAGGGATTCCCCCAGACCAGCAGCGTTTGATTTTCGCCGGAAAGCAGCTTGAGGATCGTCGTACTCTTCCCGACTACAACATCCAGAAGGAGTCAACTCTCCATCTCGTGCTCCGTCTCCGTGGTGGTGGATCCATGGACCTCCATCTAATTTTCGGTCCAACTTGCACAGGAAAGACGACGACCGCGATAGCTCTTGCCCAGCAGACAGGGCTTCCAGTCCTTTCGCTTGATCGGGTCCAATGCTGTCCTCAACTATCAACCGGAAGCGGACGACCAACAGTGGAAGAACTGAAAGGAACGACGCGTCTCTACCTTGATGATCGGCCTCTGGTGGAGGGTATCATCGCAGCCAGCAAGCTCATCATAGGCTGATCGAGGAGGTGTATAATCATGAGGCCAACGGCGGGCTTATTCTTGAGGGAGGATCCACCTCGTTGCTCAACTGCATGGCGCGAAACACCTATTTGGAGTGCAGATTTTCGTTGGCATATTATTCGCCACAAGTTACCCGACCAAGAGACCTTCATGAAAGCGGCCAAGGCCAGAGTTAAGCAGATGTTGCACCCCGCTGCAGGCCATTCTATTATTCAAGAGTTGCTTTATCTTTGGAATGAACCTCGGCTGAGGCCCATTCTGAAACAGATCGATGGATATCGATATGCCATGTTGTTTGCTAGCCAGAACCAGATCACGGCAGATATGCTATTGCAGCTTGACGCAATATGGAAGGTAAGTTGATTAATGGGATCGCTCACGAGTATTTCATCCATGCGCGCCAACAGGAACAGAAATTCCCCCAAGTTAACGCAGCCGCTTTCGACGGATTCGAAGGTCATCCGTTCGGAATGTATTAGGTTACGCCAGCCCTGCGTCGCACCTGTCTTCATCTGGATAAGATGTTCGTAATTGTTTTTGGCTTTGTCCTGTTGTGGCAGGGCGGCAAATACTTCCGACAATCCATCGTGTCTTCAAACTTTATGCTGGTGAACAAGTCTTAGTTTCCACGAAAGTATTATGTTAAATTTTAAAATTTCGATGTATAATGTGGCTATAATTGTAAAAATAAACTATCGTAAGTGTGCGTGTTATGTATAATTTGTCTAAATGTTTAATATATATCATAGAACGCAATAAATATTAAATATAGCGCTTTTATGAAATATAAATACATCATTACAAGTTGTTTATATTTCGGGTGGACTAGTTTTTAATGTTTAGCAAATGTCCTATCAGTTTCTCTTTTGTCGAACGGTAATTTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGGAATTC SEQID4AGCTTTGGCAGGATATATACCGGTGTAAACGAAGTGTGTGTGGTTGATCCAAAATCTATCGTACCTTTAGAAAGTGTAGCTATGAAGGATAGTCTCACTTATGAAGAACTACCTATTGAGATTCTTGATCGTCAGGTCCGAAGGTTGAGAAAAATAGAAGTCGCTTCAGTTACGGCTTTGTGGAGGAGTAAGGGTACCTACTCTAGAATCGAGCTCATCGTTATGCTATAAATTTCATATATTTAGTTGGGAGTAGGCTTTATACCGAGTTGGACTACGGTCAGTCACTTTCAAGTCCTAGAACTACGTGCCCCTGTAGGTTATAAGTCTCCTCTGTGGGCATCAATTTAGTGATCATGCCAGTCATGCCTCTATACCTCTGACAGGATATATGGTACTGTAAACACTAGTTGTCAATAAGTCGCTGTGTATGTTTGTTTGAGATCTCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGGTCACCTATCTCGAGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTCCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTCGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTCCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCCTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTCATGTGTATTTCAGTAAAACAGCTTGCCTCATCCGGTCGCTGCCTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTCCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGCCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGCCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACCCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGCAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGCAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCCCGCAGCCCGCCCAGCGGCAACCCATCGAGGCAGAAGCACGCCCCGGTGAATCCTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGCCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTCCGGACGTACTCAAGTTCTGCCGCCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACCTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCCGCCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCCAGATCACAGAAGGCAAGAACCCGGACCTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGACGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCCCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATACCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCCTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGCCCACGCAATCTACCAGGCCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATCACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTCTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACACATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGCGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCCACCCTGCCGCTTACCGGATACCTCTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCCTTTAAAAAATCATACAGCTCGCGCGGATCTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTATCGACGGAGCCGATTTGAAACCGCGGATCCTGCAGCCAAAGCACATACTTATCGATTTAAATTTCATCGAAGAGATTAATATCGAATAATCATATACATACTTTAAATACATAACAAATTTTAAATACATATATCTGGTATATAATTAATTTTTTAAAGTCATGAAGTATGTATCAAATACACATATGGAAAAAATTAACTATTCATAATAAAAAATAGAAAAGATACATCTAGTGAAATTAGGTGCATGTATCAAATACATTAGGAAAGGGCATATATCTGATCTAGATAATTAACGATTTTGATTTATGTATAATTTCCAAATGAAGGTTTATATCTACTTCAGAAATAACAATATACTTTTATCAGAACATTCAACAAAGTAACAACCAACTAGAGTGAAAAATACACATTGTTCTCTAAACATACAAAATTGACAAAAGAATCTCAAAATTTAGAGAACAAATCTGAATTTCTAGAAGAAAAAAATAATTATGCACTTTGCTATTGCTCGAAAAATAAATGAAAGAAATTAGACTTTTTTAAAAGATGTTAGACTAGATATACTCAAAAGCTATCAAAGGAGTAATATTCTTCTTACATTAAGTATTTTAGTTACAGTCCTGTAATTAAAGACACATTTTAGATTGTATCTAAACTTAAATGTATCTAGAATACATATATTTGAATGCATCATATACATGTATCCGACACACCAATTCTCATAAAAAGCGTAATATCCTAAACTAATTTATCCTTCAAGTCAACTTAAGCCCAATATACATTTTCATCTCTAAAGGCCCAAGTGGCACAAAATGTCAGGCCCAATTACGAAGAAAAGGGCTTGTAAAACCCTAATAAGTGGCACTGGCACAGCFTACACTCTCATTCCATCAACAAAGAAACCCTAAAAGCCGCAGCGCCACTGATTTCTCTCCTCCAGGCGAAGATGCAGATCTTCGTGAAGACCCTAACGGGGAAGACGATCACCCTAGAGGTTGAGTCTTCCGACACCATCGACAATGTCAAAGCCAAGATCCAGGACAAGGAAGGGATTCCCCCAGACCAGCAGCGTTTGATTTTCGCCGGAAAGCAGCTTGAGGATGGTCGTACTCTTGCCGACTACAACATCCAGAAGGAGTCAACTCTCCATCTCGTGCTCCGTCTCCGTGGTGGTGGATCCATGGACCTGCATCTAATTTTCGGTCCAACTTGCACACGAAAGACGACGACCGCGATAGCTCTTGCCCAGCAGACAGGGCTTCCAGTCCTTTCGCTTGATCGGGTCCAATGCTGTCCTCAACTATCAACCGGAAGCGGACGACCAACAGTGGAAGAACTGAAAGGAACGACGCGTCTCTACCTTGATGATCGGCCTCTGGTGGAGGGTATCATCGCAGCCAAGCAAGCTCATCATAGGCTGATCGAGGAGGTGTATAATCATGAGGCCAACGGCGGGCTTATTCTTGAGGGAGGATCCACCTCGTTGCTCAACTGCATGGCGCGAAACAGCTATTGGAGTGCAGATTTTCGTTGGCATATTATTCGCCACAAGTTACCCGACCAAGAGACCTTCATGAAAGCGGCCAAGGCCAGAGTTAAGCAGATGTTGCACCCCGCTGCAGGCCATTCTATTATTCAAGAGTTGGTTTATCTTTGGAATGAACCTCGGCTGAGGCCCATTCTGAAAGAGATCGATGGATATCGATATGCCATGTTGTTTGCTAGCCAGAACCAGATCACGGCAGATATGCTATTGCAGCTTGACGCAAATATGGAAGGTAAGTTGATTAATGGGATCGCTCAGGAGTATCATCCATGCGCGCCAACAGGAACAGAAATTCCCCCAAGTTAACGCAGCCGCTTTCGACGGATTCGAAGGTCATCCGTTCGGAATCTATTAGGTTACGCCAGCCCTGCGTCGCACCTGTCTTCATCTGGATAAGATGTTCGTAATTGTTTTTGGCTTTGTCCTGTGTGGCAGGGCGGCAAATACTTCCGACAATCCATCGTGTCTTCAAACTTTATGCTGGTGAACAAGTCTTAGTTTCCACGAAAGTATTATGTTAAATTTTAAAATTTCGATGTATAATGTGGCTATAATTGTAAAAATAAACTATCGTAAGTGTGCGTGTTATGTATAATTTGTCTAAATGTTTAATATATATCATAGAACGCAATAAATATTAAATATAGCGCTTTTATGAAATATAAATACATCATTACAAGTTGTTTATATTTCGGGTGGACTAGTTTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACCGTAATTTAGAGTGTTTTTTTTCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGACTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTTTAGTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGGAATTCTATCCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTG GCTGGCTGGA SEQID5ATGAGAAATTTATTCCCCATATTGATGCTAATCACCAATTTGCCACTCAACAACGATAACAACAACAACAACAACAACAACAATAATTATAATCTCATACACGCAACGTGTAGGGAGACCCCATATTACTCCCTATGTCTCACCACCCTACAATCCGGTCCACGTAGTAACGAGGTTGAGGGTGGTGATGCCATCACCACCCTAGGCCTCATCATGGTGGACGCCGTGAAATCAAACTCCATAGAAATAATGGAAAAAATAAAAGAGCTAGAGAAATCGAACCCTGAGTGGCGGGCCCCACTTAGCCAGTGTTACGTGGCGTATAATGCCGTCCTACGAGCCGATGTAACGGTAGCCGTTGAAGCCTTAAAGAAGGGTGCCCCCAAATTTGCTGAAGATGGTATGGATGATGTTGTTGCTGAAGCACAAACTTGTGAGTATAGTTTTAATTATTATAATAAATTGGATTTTCCAATTTCTAATTTGAGTAGGGAAATAATTGAACTATCAAAAGTTGCTAAATCCATAATTAGAATGTTATTATGA SEQID6GAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTACTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTCTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTGTTTTTGTTCATCTGTAG CTTGGTAG SEQID7TTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAA TAG SEQID8ACCTTATTTCACTACCACTTTCCACTCTCCAATCCCCATACTCTCTGCTCCAATCTTCATTTTGCTTCGTGAATTCATCTTCATCGAATTTCTCGACGCTTCTTCGCTAATTTCCTCGTTACTTCACTAAAAATCGACGTTTCTAGCTGAACTTGAGTGAATTAAGCCAGTGGGAGGAT SEQID9GTTAGAAATCTTCTCTATTTTGGTTTTTGTCTGTTTAGTTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTCGTTGTTCTGATTGTTT TTATGAATTTGCAGSEQID10 GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTACATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGCTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTCAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCACCTTATTTCACTACCACTTTCCACTCTCCAATCCCCATACTCTCTGCTCCAATCTTCATTTTGCTTCGTGAATTCATCTTCATCGAATTTCTCGACGCTTCTTCGCTAATTTCCTCGTTACTTCACTAGAAATCGACGTTTCTAGCTGAACTTGAGTGAATTAAGCCAGTGGGACGATGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGCTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTCTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCATCCTCCCACTGGCTTAATTCACTCAAGTTCAGCTAGAAACGTCGATTTCTAGTGAAGTAACGAGGAAATTAGCGAAGAAGCGTCGAGAAATTCGATGAAGATGAATTCACGAAGCAAAATGAAGATTGGAGCAGAGAGTATGGGGATTGGAGAGTGGAAAGTGGTAGTGAAATAAGGTAACCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAACTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA SEQID11GTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTGTTTCATATCTCATCTCATCTATCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAG SEQID12GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCACCTTATTTCACTACCACTCCACTCTCCAATCCCCATACTCTCTGCTCCAATCTTCATTTTGCTTCGTGAATTCATCTTCATCGAATTTCTCGACGCTTCTTCGCTAATTTCCTCGTTACTTCACTAGAAATCGACGTTTCTAGCTGAACTTGAGTGAATTAAGCCAGTGGGAGGATGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCATCCTCCCACTGGCTTAATTCACTCAAGTTCAGCTAGAAACGTCGATTCTAGTGAAGTAACGAGGAAATTAGCGAAGAAGCGTCGAGAAATTCGATGAAGATGAATTCACGAAGCAAAATGAACATTGGAGCAGAGAGTATGGGGATTGGAGAGTGGAAAGTGGTAGTGAAATAAGGTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAG A SEQID13GAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCAC SEQID14GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAGAAGGAATTCAAGGTTAGAAATCTTCTCTATTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGGTGAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAGAATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTCAGTATAATATAGTAAAAAAATAGTCTAGA SEQID15GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATCAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAGGAATTCGTGGTAACTTTTACTCATCTCCTCCATTAATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGCTGAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAGAATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAA AATAGTCTAGA SEQID16TCATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATACGGAGAAATGTGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAG SEQID17GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAGGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTCTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAACGTGAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAGAATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA SEQID18GGTACCGAACCATCCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCCCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTCTTCAGAGAAGACCACAGATGTCATCATATTCTCATTGTATCACATCTGTGACCACTTACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGCGAGAAATGTGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAGGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGGTGAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAGTGGTCACACATCTGATACAATGAGAATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTACCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATCGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAA AAATAGTCTAGA SEQID19GCTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTACATATTATCTTTTATTACTATAATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGCGCCCCATTGCAAGCCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTCTCGATGACCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTCACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTCCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCACGGATCCTCATATTCTAGTTGTATGTTGTTCAGAGAACACCACAGATGTGATCATATTCTCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGCAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGAAAAACTTTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAGGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATACCCCTTAATTTTGAGTTTTTTTTCGCTTGTTTTGATGCAAAAGGCCTAAAATTTGACTTTTTTTACGTTGGTTTGATGGAAAAGCCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTCAGTTTTTCCGGTTGATTTCATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCACATGGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTCTCCTCCAATAGCTGAAGGTGAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAACTGGTCACAGATCTGATACAATGACAATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATCCAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA SEQID20GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGCAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCCTGAATCAACAAAGAGAGGCCCCATAATACTGTCGATGAGCACCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAACCTGCTACAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTCATATTCTAGTTGTATGTTGTTCAGAGAAGACCACAGATGTGATCATATCTCATTGTATCAGATCTGTGACCACTTACCTGATACCTCCCATGAAGTTACCTGTATGATTATACGTGATCCAAAGCCATCACATCATGTTCACCTTCAGCTATTGGAGGAGAAGTGAGAAGTAGGAATTGCAATATGAGGAATAATAAGAAAAACTGTAAAAGCTAAATTAGCTGGGTATGATATAGGGAGAAATGTGTAAACATTGTACTATATATAGTATATACACACGCATTATGTATTGCATTATGCACTGAATAATACCGCAGCATCAAAGAAGGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTTGTTTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGCGGTATTATTCAGTGCATAATGCAATACATAATGCGTGTGTATATACTATATATAGTACAATGTTTACACATTTCTCCCTATATCATACCCAGCTAATTTAGCTTTTACAAAGTTTTTCTTATTATTCCTCATATTGCAATTCCTACTTCTCACTTTCTCCTCCAATAGCTGAAGGTGAACATGATGTGATGGCTTTGGATCACGTATAATCATACAGGTAACTTCATGGGAGGTATCAGGTAAGTGGTCACAGATCTGATACAATGAGAATATGATCACATCTGTGGTCTTCTCTGAACAACATACAACTAGAATATGAAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATA GTCTAGA SEQID21TTAGAGTGTGGGTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAATCAATAATTGACTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGATTTGCATAGAGAGCACAAA SEQID22GAGGGGGAAGTGAATGAAAAATAACAAAGGCACAGTAAGTAGTTTCTCTTTTTATCATGTGATGAAGGTATATAATGTATGTGTAAGAGGATGATGTTATTACCACATAATAAGAGATGAAGAGTCTCATTTTCTGCTTAAAAAAACAAT TCACTGGC SEQID23GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCGAGTCTGGGTAAGTAATTAAGTTTAGGGATTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTGACATAATCCACAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAATCAATAATTGACTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGATTTGCATAGAGAGCACAGAATTCAAGGTTAGAAATCTTTCTCTATTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTCCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCACATGGATATCTGTGCTCTCTATGCAAATCTAGCTTTTCGAATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGATGAAGATTGGAGAAGTCAATTATTGATTCACACACAGGAATTAAGTGTGTTGTGTTGCGTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTATTTATCAATAAAAGCACGAAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATTTGTCCATCCCACAAATCCCTAACTTAATTACTTACCCACACTCTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAAT AGTCTAGA SEQID24GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGACAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCGAGTGTGGGTAAGTAATTAAGTAGGGATTTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAATCAATAATTGACTTCTCCAATCTTCATCAATAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGATTTGCATAGACAGCACAGAATTCGTGGTAACTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTCATATCTCATCTCATCTATTTCTGATTTGATTTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCTGTGCTCTCTATGCAAATCTAGCTTTTCGAATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGATGAAGATTGGAGAAGTCAATTATTGATTCACACACAGGAATTAAGTGTGTTGTGTTGCGTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTATTTATCAATAAAAGCACGAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATGTCCATCCCACAAATCCCTAACTTAATTACTTACCCACACTCTAAGCTTTTGATTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGACTTTTTTTTGCTATATGGATTTCGTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATA TAGTAAAAAAATAGTCTAGASEQID25 GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTCGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATCCCTATAATACAGTGTCCACAGTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTACACCACACATCACCGATCCGACTGTGGGTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAATATAAGAGAGTGCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAATCAATAATTGACTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGATGCATAGAGAGCACAGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTACATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTCTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCTGTGCTCTCTATGCAATCTAGCTTCGAATGAGAGTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGATGAAGATTGGAGAAGTCAATTATTGATTCACACACAGGAATTAAGTGTGTGTGTTGCCTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTATTTATCAATAAAAGCACGAAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATTTGTCCATTTCCCACAAATCCCTAACTTAATTACTTACCCACACTCTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCCATAAATATGCTTCTCAGGAATTTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA SEQID26GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGCACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAACAGAGCGCCCATAATACTGTCCATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAACTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCACGGATCCGAGTGTGGGTAAGTAATTAAGTTAGGGATTTGTGGGAAATGGACAAATATAAGAGAGTCCAGGGGAGTAGTGCAGGAGATTTTCGTGCTTTTATTGATAAATAAAAAAAGGGTGACATTTAATTTCCACAAGAGGACGCAACACAACACACTTAATTCCTGTGTGTGAATCAATAATTGACTTCTCCAATCTTCATCAATAAAATAATTCACAATCCTCACTCTCTTATCACTCTCATTCGAAAAGCTAGATTTGCATAGAGAGCACAGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTCTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTCCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCTGTGCTCTCTATGCAAATCTAGCTTTTCGAATGAGACTGATAAGAGAGTGAGGATTGTGAATTATTTTATTGATGAAGATTGGAGAAGTCAATTGATTCACACACAGGAATTAAGTGTGTTGTGTTGCGTCCTCTTGTGGAAATTAAATGTCACCCTTTTTTTATTTATCAATAAAAGCACGAAAATCTCCTGCACTACTCCCCTGCACTCTCTTATATTTGTCCATTTCCCACAAATCCCTAACTTAATTACTTACCCACACTCTAAGCTTTTGATTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTITCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATCCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATA GTCTAGA SEQID27ATGGCAAGCTTGTGCAATAGTAGTAGTACATCTCTCAAACTCCTTTTACTTCTTCCTCCACTTCTTTATCTTCCACTCCTAAGCCCTCTCAACTTTTCATCCATGGAAAACGTAACCAAATGTTCAAAGTTTCATGCAAGGTTATCAATAATAACCGTGACCAAAACGTTGAAACGAATTCTGTTCATCGAAGAAATGTTCTTCTTGGCTTAGGTGGTCTTTATGGTGTTGCTAATGCTATACCATTAGCTGCATCCGCTGCTCCAACTCCACCTCCTGATCTCTCGTCTTGTAGTATAGCCAGGATTAACGAAAATCAGGTGGTGCCGTACAGTTGTTGCGCGCCTAAGCCTGATGATATGGAGAAAGTTCCGTATTACAAGTTCCCTTCTATGACTAAGCTCCGTGTCCGTCAGCCTGCTCATGAAGCTAATGAGGAGTATATTGCCAAGTACAATCTGGCGATTAGTCGAATGAGAGATCTTGATAAGACACAACCTTTAAACCCTATTGGTTTTAAGCAACAAGCTAATATACATTGTGCTTATTGTAATGGTGCTTATAGAATTGGTGGCAAAGAGTTACAAGTTCATAATTCTTGGCTTTTCTTCCCGTTCCATAGATGGTACTTGTACTTCCACGAGAGAATCGTGGGAAAATTCATTGATGATCCAACTTTCGCTTTGCCATATGGAATTGGGACCATCCAAAGCGTATGCGTTTTCCTGCCATGTATGATCGTGAAGGGACTTCCCTTTTCCATGTAACACGTGACCAAAGTCACCGAAATGGAGCAGTAATCGATCTTGGTTTTTTCGGCAATGAAGTCGAAACAACTCAACTCCAGTTGATGAGCAATAATTTAACACTAATGTACCGTCAAATGGTAACTAATGCTCCATGTCCTCGCATGTTCTTTGGTGGGCCTTATGATCTCGGGATTAACACTGAACTCCCGGGAACTATAGAAAACATTCCTCACGGTCCTGTCCACATCTGGTCTGGTACAGTGAGAGGTTCAACTTTGCCCAATGGTGCAATATCAAACGGTGAGAATATGGGTCATTTTTACTCAGCTCCTTTGGACCCGGTTTTCTTTTGCCATCACAGCAATGTGGATCGGATGTGGAGCGAATGGAAAGCGACAGGAGGGAAAAGAACAGATATCACACATAAAGGTTGGTTGAACTCCGAGTTCTTTTTCTATGATGAAAATGAAAACCCTTACCGTGTGAAAGTCCGAGACTGTTTGGACACGAAGAAGATGGGGTATGATTATGCACCAATGGCCACCCCGTGGCGTAACTTCAAGCCAATAACAAAAACTACAGCTGGGAAAGTGAATACAGCTTCTCTTCCGCCAGCTAGCAATGTATTCCCAGTGGCTAAACTCGACAAAGCAATTTCGTTTTCCATCAATAGGCCGACTTCGTCAAGGACTCAACAAGAGAAAAATGCACAAGAGGAGATGTTGACATTCAGTAGCATAAGATATGATAACAGAGGGTACATAAGGTTCGATGTGTTCCTGAACGTGGACAATAATGTGAATGCGAATGAGCTTGACAAGGCGGAGTTTGCGGGGAGTTATACTAGTTTGCCACATGTTCATAGAGCTGGTGAGACTAATCATATCGCGACTGTTGATTTCCAGCTGGCGATAACGGAACTGTTGGAGGATATTGGTTTGGAAGATGAAGATACTATTGCGGTGACTCTGGTGCCAAAGAGAGGTGGTGAAGGTATCTCCATTGAAAGTGCGACGATCAGTCTTGCAGATTGTTAA SEQID28ATGGCAAGCTTGTGCAATAGTAGTAGTACATCTCTCAAAACTCCTTTTACTTCTTCCTCCACTTTCTTTATCTTCCACTCCTAAGCCCTCTCAACTTTTCATCCATGGAAAACGTAACCAAATGTTTCAAAGTTTCATGCAAGGTTATCAATAATAACGGTGACCAAAACGTTGAAACGAATTCTGTTGATCGAAGAAATGTTCTTCTTGGCTTAGGTGGTCTTTATGGTGTTGCTAATGCTATACCATTAGCTGCATCCGCTGCTCCAACTCCACCTCCTGATCTCTCGTCTTGTAGTATAGCCAGGATTAACGAAAATCAGGTGGTGCCGTACAGTTGTTGCGCGCCTAAGCCTGATGATATGGAGAAAGTTCCGTATTACAAGTTCCCTTCTATGACTAAGCTCCGTGTCCGTCAGCCTGCTCATGAACCTAATGAGGAGTATATGCCAAGTACAATCTGGCGATTAGTCGAATGAGAGATCTTGATAAGACACAACCTTTAAACCCTATTGGTTTTAAGCAACAAGCTAATATACAGTGGGCTTATGGTAATGGTGCTTATAGAATTGGTGGCAAAGAGTTACAAGTTCATAATTCTTGGCTTTTCTTCCCGTCCATAGATGGTACTTGTACTTCCACGAGAGAATCGTGGGAATCATTGATGATCCAACTTTCGCTTTGCCATATTGGAATTGGGACCATCCAAAGGGTATCCGTTCCTGCCATGTATGATCGTGAACGCACTTCCCTTTTCGATGTAACACGTGACCAAAGTCACCGAAATGGAGCAGTAATCGATCTTGGTTTTTTCGGCAATGAAGTCGAAACAACTCAACTCCAGTTGATGAGCAATAATTTAACACTAATGTACCGTCAAATGGTAACTAATGCTCCATGTCCTCGGATGTTCTTTGGTGGGCCTTATGATCTCGCGATTAACACTGAACTCCCGGGACTATAGGAAACATTCCTCTCGGTCCTGTCCACATCTGGTCTGGTACAGTCAGAGGTTCAACTTTGCCCAATGGTGCAATATCAAACGGTGAGAATATGGGTCATTTACTCAGCTGCTTTGGACCCGGTTTTCTTTTGCCATCACAGCAATGTGGATCGGATGTGGAGCGAATGGAAAGCGACAGGAGGGAAAAGAACACATATCACACATAAAGGTTGGTTGAACTCCGAGTTCTTTTTCTATGATGAAAATGAAAACCCTTACCGTGTGAAAGTCCGAGACTGTTTGGACACGAAGAAGATGGGGTATGATTATGCACCAATGGCCACCCCGTGGCGTAACTTCAAGCCAATAACAAAAACTACAGCTGGGAAAGTGAATACAGCTTCTCTTCCGCCAGCTAGCAATGTATTCCCAGTGGCTAAACTCGACAAAGCAATTTCGTTTTCCATCAATAGGCCGACTTCGTCAAGGACTCAACAAGAGAAAAATGCACAAGAGGAGATGTTGACATTCAGTAGCATAAGATATGATAACAGAGGGTACATAGGTTCGATGTGTTCCTCAACGTGGACAATAATGTGAATGCGAATGAGCTTGACAAGCCGGAGITTGCGGGGAGTTATACTAGTTTGCCACATGTTCATAGAGCTGGTGAGACTAATCATATCGCGACTGTTGATTTCCAGCTCGCGATAACGGAACTGTTGGAGGATATTGCTTTGGAAGATGAAGATACTATTGCGGTGACTCTGGTGCCAAAGAGAGGTGGTGAAGGTATCTCCATTGAAAGTGCGACGATCAGTCTTGCAGATTGTTAA SEQID29TTAGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTTTGTTGCTTGATTTCATTGAAGTTGTTATTCAAGAATAATCAGTTACAATTATGTTT GGG SEQID30GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCACTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGCAGGGAGTTGGTTCAGTTTTTTAGATACTAGCAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTTAGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTTTGTTGCTTGATTTCATTCAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTCCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCCGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACATTTGTTTGATGAAAAAGGCCTTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCCGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTTGAGTTTTTTCCCCGTGTTTTAGATTCTTTGGTTTTAATTCTCCAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGAGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTGATGCTGATCCATAATTGTAACTGATTTATTCTTGAATAACAACTTCAATGAAATCAACCAACAAAGCTGATTTCAACAGAGGAAAAAACAGAACAAGAAAACAAAAACAGAGCATCATCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGCATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA SEQID31GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGCCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAAGAAGGATCCTTAGTCTCTATTGAATCTGCTGACATTACACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTTTGTTGCTTGATTTCATTGAAGTGTTATTCAAGAATAAATCAGTTACAACAATTATGGAATTCGTGGTAACTTTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGATCCATAATTGTAACTGATTTATTCTTGAATAACAACTTCAATGAAATCAAGCAACAAAGCTGATTTCAACAGAGGAAAAAACAGAACAAGAAAACAAAAACAGAGCATCATCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTCTCCAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATCTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAA AATAGTCTAGA SEQID32GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTTTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTGACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTAGACACATCACGGATCCTTAGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTTTGTTGCTTGATTTCATTGAAGTTGTTATTCAAGAATAAATCAGTTACAATTATGGAATTCAAGGTTAGAAATCTTCTCTATTTTTGGTTTTTGTCTGTTTAGATTCTCGAATTAGCTAATCAGGTGCTGTTATAGCCCTTAATTTTGAGTTTTTTTTCGGTTGTTTTGATGGAAAAGGCCTAAAATTTGAGTTTTTTTACGTTGGTTTGATGGAAAAGGCCTACAATTGGAGTTTTCCCCGTTGTTTTGATGAAAAAGCCCCTAGTTTGAGATTTTTTTTCTGTCGATTCGATTCTAAAGGTTTAAAATTAGAGTTTTTACTTTGTTTGATGAAAAAGGCCTAAATTTGAGTTTTTCCGGTTGATTTGATGAAAAAGCCCTAGAATTTGTGTTTTTTCGTCGGTTTGATTCTGAAGGCCTAAAATTTGAGTTTCTCCGGCTGTTTTGATGAAAAAGCCCTAAATTGAGTTTCTCCGGCTGTTTTCATGAAAAAGCCCTAAATTGAGTTTTTTCCCCGTGTTTTAGATTGTTTGGTTTTAATTCTCGAATCAGCTAATCAGGGAGTGTGAAAAGCCCTAAAATTTGSGTTTTTTTCGTTGTTCTGATTGTTGTTTTTATGAATTTGCAGATGGATATCCTTCTTTGATGCTGATCCATAATTGTAACTGATTTATTCTTGAATAACAACTTCAATGAAATCAAGCAACAAAGCTGATTTCAACAGAGGAAAAAACAGAACAAGAAAACAAAAACAGAGCATCATCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGCAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAG TCTAGA SEQID33GGTACCGAACCATGCATCTCAATCTTAATACTAAAAAATGCAACAAAATTCTAGTGGAGGGACCAGTACCAGTACATTAGATATTATCTTTTATTACTATAATAATATTAATTAACACGAGACATAGGAATGTCAAGTGGTAGCGGTAGGAGGGAGTTGGTTCAGTTTTTTAGATACTAGGAGACAGAACCGGAGGGGCCCATTGCAAGGCCCAAGTTGAAGTCCAGCCGTGAATCAACAAAGAGAGGGCCCATAATACTGTCGATGAGCATTCCCTATAATACAGTGTCCACAGTTGCCTTCCGCTAAGGGATAGCCACCCGCTATTCTCTTCACACGTGTCACTGAAACCTGCTACAAATAAGGCAGGCACCTCCTCATTCTCACACTCACTCACTCACACAGCTCAACAAGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATGAATTTCTGTTTTGTTTTTGTTCATCTGTAGCTTGGTAGATTCCCCTTTTTGTAGACCACACATCACGGATCCTTAGTCTCTATTGAATCTGCTGAGATTACACTTTGATGGATGATGCTCTGTTTTTGTTTTCTTGTTCTGTTTTTTCCTCTGTTGAAATCAGCTTTGTTGCTTGATTTCATTGAAGTTGTTTATTCAAGAATAAATCAGTTACAATTATGGAATTCGTGGTAACTTTTACTCATCTCCTCCAATTATTTCTGATTTCATGCATGTTTCCCTACATTCTATTATGAATCGTGTTATGGTGTATAAACGTTGTTTCATATCTCATCTCATCTATTCTGATTTTGATTCTCTTGCCTACTGAATTTGACCCTACTGTAATCGGTGATAAATGTGAATGCTTCCTCTTCTTCTTCTTCTTCTCAGAAATCAATTTCTGTTTGTTTTTGTTCATCTGTAGCTTGATATCCTTCTTTGATGCTGATCCATAATTGTAACTGATTTATTCTTGAATAACAACTTCAATGAAATCAAGCAACAAAGCTGATTTCAACAGAGGAAAAAACAGAACAAGAAAACAAAAACAGAGCATCATCCATCAAAGTGTAATCTCAGCAGATTCAATAGAGACTAAGCTTTTGATTTTAATGTTTAGCAAATGTCCTATCAGTTTTCTCTTTTTGTCGAACGGTAATTTAGAGTTTTTTTTGCTATATGGATTTTCGTTTTTGATGTATGTGACAACCCTCGGGATTGTTGATTTATTTCAAAACTAAGAGTTTTTGCTTATTGTTCTCGTCTATTTTGGATATCAATCTTAGTTTTATATCTTTTCTAGTTCTCTACGTGTTAAATGTTCAACACACTAGCAATTTGGCTGCAGCGTATGGATTATGGAACTATCAAGTCTGTGGGATCGATAAATATGCTTCTCAGGAATTTGAGATTTTACAGTCTTTATGCTCATTGGGTTGAGTATAATATAGTAAAAAAATAGTCTAGA SEQID34GTCCATGATGTCTTCAGGGTGGTAGCATTGACTGATGGCATCATAGTTTTTTTTTTAAAAGTATTTCCTCTATGCATATTATTAGTATCCAATAAATTTACTGGTTCTTGTACATAGAAAAAGTGCATTTGCATGTATGTGTTTCTCTGAAATTTTCCCCAGTTTTTGGTGCTTTGCCTTTGGAGCCAAGTCTCTATATGTATAAGAAAACTAAGAACAATCACATATATCAAATATTAG SEQID35ACGAACTTGTGATCGCGTTGAAAGATGAACGCTACATAGAGCTTCTTGACGTATCTGGCAATATTGCATCAGTCTTGCCGGAATTTCATGTGACAACAAGGTTTGCAATTCTTTCCACTATTAGTAGTGCAACGATATACGCAGAGATGAAGTGCTGAACAAACATATGTAAAATCGATGAATTTATGTCGAATGCTGGGACGGGCTTCAGCAGGTTTTGCTTAGT SEQID36CCGCGGTTTTCTCTCCATCGCGTCAGAGGCCGGTTTTCGTCGGCATCGAAGAGGGCCACTCGTTTACCGTCATTTGCCAAAGCAGCGCAAAGGCCCATGAGTGCGGTGGTTTTGCCAGCACCCCCTTTGAAAGAGCAAAACGTCAAAAGTTGCATATTCTGATCCCGCCTGTCCTGTGAAACGGAGTGCATTTGTATTTTTGTTCGTATAAATGTTTTTGTGATTATCGATGAGTAAAAGCGTTGTTACACTATTTTTTATTTCAAATTCGTTATAATTAAATTGCAATTGTAGCAATTATATTCGGTTTTTCCTGTAAATATACTGTTGATTTCATATCGAGTAGGGCTAGACTTTAATCTGTCTACCCGGGCACATTTCGTGCTGGAGTATTCAGACCTTCCGCTTTTTTTGGAGGAAGCTATGTCAAAACACACCAGAGTCACGTCGAGTGAGACTGCCATCAACCAGCATCGATCCCTGAACGTTGAAGGGTTTTTAACGTCGTGAGTGCCCGTCTGCGATCGGCCGAGTATGAAACCTTTTCCTATCAAGCGCGCCTGCTGGGACTCGGATAGTATGGCAATTCGCGTTGCGGTGCGTCGCATCGGGGGCTTTCTCGAAATAGATGCACACACCCGAGAAAACATGGAAGCCATACTTCAGTCCATCGGAATACTCTCAAGTAATGTATCCATGCTTCTATCTCCCTACGCCGAAGACCCTCGATCGGATCTGGAGGCTGTGCGAGATGAACGTATTGCTTTTGGTGAGGCTTTCGCCGCCCTCGATGGCCTACTCCGCTCCATTTTGTCCGTATCCCGGCGACGGATCGACGGTTGCTCGCTATTGAAAGGTGCCTTGTACGACTTGACCACGCACCTGACGGGAGAAAATTGGATGCCCGATCGCGCTCAAGTAATCATTCGCATTGTGCCAGGAGGTGGAACCAAGACCCTTCAGCAGATAATCAATCAGTTGGAGTACCTGTCCCGTAAGGGAAAGCTGGAACTGCAGCGTTCAGCCCGGCATCTCGATATTCCCGTTCCGCCGGATCAAATCCGTGAGCTTGCCCAAAGCTGGGTTACGGAGGCCGGGATTTATGACGAAAGTCAGTCAGACGATGATAGGCAACAAGACTTAACAACACACATTATTGTAAGCTTCCCCGCAGGTACCGACCAAACCGCAGCTTATGAAGCCAGCCGGGAATGGGCAGCCGAGATGTTTGGGTCAGGATACGGGGGTGGCCGCTATAACTATCTGACAGCCTACCACGTCGACCGCGATCATCCACATTTACATGTCGTGGTCAATCGTCGGGAACTTCTGGGGCACGGGTGGCTGAAAATATCCAGGCCCCATCCCCAGCTGAATTATGACGGCTTACGGAAAAAGATGGCAGAGATTTCACTTCGTCACGGCATAGTCCTGGATGCGACTTCGCGAGCAGAAAGGGGAATAGCAGAGCGACCAATCACATATGCTGAACATCGCCGCCTTGAGCGGATGCAGGCTCAAAAGATTCAATTCGAAGATACAGATTTTGATGAGACCTCGCCTGAGGAAGATCGTCGGGACCTCAGTCAATCGTTCGATCCATTTCGATCGGACCCATCTACCGGCGAACCGGACCGTGCAACCCGACATGACAAACAACCGCTTGAACAGCACGCCCGTTTCCAGGAGTCCCCCGGCTCCAGCATCAAACCCGACGCACGGATCCGCGTATCATTGGAGAGCGAGCGGAGTGCCCAACCATCCGCGTCCAAAATCCCTGTAATTGGGCATTTCGGGATTGAGACTTCCTATGTCGCTGAAGCCAGCGTGCGCAAACGAAGCGGCATTTTCGGTACTTCTCGCCCGGTGACTGACGTTGCCATGCACACAGTCAAGCGCCAGCAGCGATCAAAACGACGTAATGACGAGGAGGCAGGTCCGAGCGGAGCAAACCGTAAAGGATTGAAGGCTGCGCAAGTTGATTCCGAGGCAAATGTCGGTGAGCAAGACACTCGCGATGACAGCAACAAGGCGGCTGATCCGGTGTCTGCTTCCATCGGTACCGAGCAACCGGAAGCTTCTCCAAAGCGTCCGCGTGACCGTCACGATGGAGAATTGGGTGGACGCAAACGTGCAAGAGGTAATCGTCGCTCGAGCTCGAGCGGGGGGACCTAGAGACAGGAAGGACCGAATAATGGCC GCGG SEQID37ATGGCTTCTGTGCTGGCTTCTCTGTTTCCAAAACTGGGCTCTGGGTACTTTCAGATCATGCTTCTGTTGTATCCATCAACCTCTTTGTGGCACTCCTTTGTGCTTGCATCATCATTTGGTCATCTCTTGGAGGAGAACCGCTGGGTTAATGAGTCCATTACTGCCCTCATAATTGGTTTGTGTACAGGAGTGGTTATCTTGCTCGTAAGTGGTGGAAAGAGCTCACACCTTCTGGTTTTCAGTGAAGATCTCTTTTTCATATATGTACTTCCTCCAATCATATTTAATGCAGGGTTTCAGGTAAAAAAGAAGCAATTTTTCGTAAACTTCATTACTATAATGATGTTCGGAGCCATTGGTACCCTGGTCTCATGTGCCATTATATCATTAGGTGCCATTCAAACTTTCAAGAAGTTGGACATTGAATTTCTAGATATTGGGGATTATCTTGCAATTGGAGCAATATTTGCTGCCACAGATTCCGTCTGCACATTGCAGGTCCTACATCAGGATGAGACACCCCTCCTTTACAGTCTTGTATTTGGAGAAGGAGTTGTAAATGATGCTACATCGGTGGTGCTTTTCAATGCTATTCAAAACTTCGACCTTACGAGCATGAATCCCAGTATAGCCCTCAGTTTCCTTGGCAACTTCTTCTATCTGTTCCTTGCTAGCACTTTACTGGCAGCAGGAACTGGTCTTCTTAGTGCTTACATTATCAAGAAGCTATATTTTGGCAGGCACTCCACAGATCGTGAGGTTGCCCTTATGATGCTCATGGCTTACTTATCATACTTGCTGCCCGAATTATTCTATTTGAGTGGGATTCTCACCGTCTTTTTCTGTGGTATTGTAATGTCTCACTACACTTGGCACAATGTGACCGAGAGTTCAAGAGTCACTACAAGGCACACTTGCAACTTTGTCATTTCTTGCAGAGACTTTCCTCTTCCTCTATGTCGGCATGGATGCTTTGGATATCGAGAAGTGGAAATTTGTTGGTGACAGGCCTGGATTATCAATTTCCGTGAGTTCAATACTGATGGGACTAATCTTGCTTGGGAGAGCTGCCTTTGTTTTTCCATTATCATTCTTATCCAACTTAATGAAGAAATCCTCGGAGCAAAAAATTACCTTTAGGCAGCAAGTGATAATATGGTGGGCAGGTTTGATGAGAGGCGCAGTGTCCATGGCACTGGCATATAATAAGTTCACTCGTGGGGGACACACTCAACTGCAGGACAATGCAATAATGATTACCAGCACGATAACCATTGTTCTATTCAGCACAATGGTATTCGGTTTAATGACAAAACCCCTTATAAGTCTCCTGCTGCCACCACAGAGGCAATTGAGTACAGTGTCATCAGGCGCAAATACTCCAAAGTCTCTAACAGCCCCACTCCTAGGCAGTCGAGAGCACTCTGAAGTTGATTTAAATGTTCCAGATCTTCCTCACCCACCAAGTTTGAGGATGCTACTTACCGCACCAAGTCATAAAGTGCATCGGTACTGGCGCAAGTTTGACGATGCATTCATGCGCCCTATGTTTGGTGGTCGGGGATTTGCTCCTCCTGCCCCTGGTTCTCCAACGGAACAG GGTCCATGAGGTACCAATCSEQID38 ATGGCTTCTGTGCTGGCTTCTCTGTTTCCAAAACTGGGCTCTTTGGGTACTTCAGATCATGCTTCTGTTGTATCCATCAACCTCTTTGTGGCACTCCTTTGTGCTTGCATCATCATTGGTCATCTCTTGGAGGAGAACCGCTGGGTTAATGAGTCCATTACTGCCCTCATAATTGGTTTGTGTACAGGAGTGGTTATCTTGCTCGTAAGTGGTGGAAAGAACTCACACCTTCTGGTTTTCAGTGAAGATCTCTTTTTCATATATGTACTTCCTCCAATCATATTTAATGCACGCTTTCAGGTAAAAAAGAAGCAATTTTTCGTGAACTTCATTACTATAATGATGTTCGGAGCCATTGGTACCCTGGTCTCATGTGCCATTATATCATTAGGTGCAATTCAAACTTTCAAGAAGTTGGACATTGAATTTCTAGATATTGGGGATTATCTTGCAATTGGAGCAATATTTGCTGCCACAGATTCCGTCTGCACATTGCAGGTCCTACATCAGGATGAGACACCCCTCCTTTACAGTCTTGTATTTGGAGAAGGAGTTGTAAATGATGCTACATCGGTGGTGCTTTTCAATGCTATTCAAAACTTTGACCTTACGAGCGTGAATCCCAGTATAGCCCTCAGTTTCCTTGGCAACTTCTTCTATCTGTTCCTTGCTAGCACTTTACTGGGAGCAGGAACTGGTCTTCTTAGTGCTTACATTATCAAGAAGCTGTATTTTGGCAGGCACTCCACAGATCGTGAGGTTGCCCTTATGATGCTCATGGCTTACTTATCATACATGCTGGCTGAACTATTCTATTTGAGTGGGATTCTCACTGTATTTTTCTGTGGTATTGTAATCTCTCATTACACTTGGCACAATGTGACCGAGAGTTCAAGAGTCACTACAAGGCACGCTTTTGCAACTTTGTCATTTCTTGCAGAGACTTTCCTCTTCCTCTATGTCGGCATGGATGCTTTGGATATCGAGAAGTGGAAATTTGTTGGTGACAGGCCTGGATTATCAATTTCCGTGAGTTCAATACTGATGGGATTAATCTTGCTGGGGAGAGCTGCCTTTGTTTTTCCATTATCATTCTTCTCCAACTTAATGAAGAAATCCTCGGAGCAAAAAATTACCTTTAGGCAGCAAGTGATAATATGGTGGGCAGGTTTGATGAGAGGCGCAGTGTCCATGGCACTGGCATATAATAAGTTCACTCGTGGGGGACACACTCAACTGCAGGACAATGCATAATGATTACCAGCACGATAACCATTGTTCTATTCAGCACAATGGTATTCGGTAATGACAAAACCCCTTATAAGTCTCCTGCTGCCACCACAGAGGCAATTGAGTACAGTGTCATCAGGTGCAAATACTCCAAAGTCTCTAACAGCCCCACTCCTAGGCAGTCGAGAGGACTCTGAAGTTGATTTAAATGTTCCAGATCTTCCTCACCCACCAACTTTGAGGATGCTACTTACCGCACCAAGTCATAAAGTGCATCGGTACTGGCGCAAGTTTGACGATGCATTCATGCGCCCTATGTTTGGTGGTCGGGGATTTGCTCCTCCTGCCCCTGGTTCTCCAACGGAACAGG GTCCATGAGGTACAATCSEQID39 ATGGAAAATTCGGTACCCAGGACTGTAGAAGAAGTATTCAACGATTTCAAAGGTCGTAGAGCTGGTTTAATCAAAGCACTAACTACAGATGTCGAGAAGTTTTATCAATCGTGTGATCCTGAAAAGGAGAACTTGTGTCTCTATGGGCTTCCTAATGAAACATGGGAAGTAAACCTCCCTGTAGAGGAGGTGCCTCCAGAACTTCCGGAGCCAGCATTGGGCATAAACTTCGCACGTGATGGAATGCAAGAGAAAGACTGGTTATCACTGTTGCTGTTCACAGTGATTCATGGCTGCTTTCTGTTGCATTTTACTTTGGTGCAAGGTTTGGGTTCGGCAAGAGTGAAAGGAAGAGGCTTTTCCAAATGATAAATGATCTCCCAACAGTGTTTGAAGTTGTTACCGGAGCTGCTAAACAGACACGTGATCCCCCTCACAACAATAGCAACAAAAGCAAATCAAGTGGAAAGCCTCGACAGCCAGAGTCCCAACTCAAGGCAGTAAAGGTGTCTCCACCTAAAATGGAGAACGACAGTGGGGAGGAGGAAGAAGAAGAAGAGGATGAACAAGGAGCAACTCTCTGTGGAGCTTGTGGTGATAATTATGCCACTGATGAATTCTGGATTTGCTGTGATATTTGTGAGAGATGGTTCCATGGCAAATGTGTGAAGATTACCCCAGCAAAAGCTGAGCATATCAAGCAGTACAAGTGTCCTAGTTGCAGTAGCAAGAGAGCTAGAGTTTAA SEQID40TGACATCTGCCAATAAAGCCAAGAATAATTGGCATTAACATGACCAAAAAAATGGTTTGGCAGCATTAAGTCAAATAAAAAAGCTACTTTAATATAAAATAATATTAAAATGCTTAATAACCAACAGTTTATAAGAAGGTTAATGTTAACATGGATGAGGAATGACCAAAAGGGGAATTATATATTAACCTTTAAATCAATCTAATTCTCTCTTTTTGTTTCTAGCTATATTTACTCGATAGATAAACTCTCTTACTTGACGAATTTTTTGATACAAGAAGACATATTTCATCATGATTTTAATTCGTCGTGTCAAATTTATTAAATAGTTTAATTTTAATCGTAAATAGATATGAAATTTAAAAAAAAATAAATATATACATATTTGAAGAATACATAAAAAGTACATATAAATCACAAATATTTAATAATTCAAGATATTAAAACACATAGAAAAATAATTACTTACAAAGAAATTCTTATTTGAATCCTCTAAATTCGAGAAGTGCAACACAAACTGAGACGAAGAAAATGAATAATATTTGATAAGAAATTTATTATAATTGAATGACCATTTAAGTAATTACGGGTAATAACAACACAATAAGGAACTGTAGTCATTTTTAATACATGGCAAGGAATATGAGAGTGTGATGAGTCTATAAATAGAAGGCTTCATTAGTGTAGAGGAGTCACAAACAAGCAATACACAATAAAATTAGTAGCTTAAACAAGATG SEQID56TTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTC SEQID94TGGCAGGATATATGAGTGTGTAAAC SEQID95 TTGGCAGGATATATCCCTCTGTAAAC

1. A method of modifying a trait of a plant, comprising (a) usingAgrobacterium-mediated transformation to stably transform plant cellswith a desired polynucleotide, wherein (i) the desired polynucleotide islocated in a vector, and (ii) the vector comprises an integration markergene in its backbone, wherein expression of the integration marker genealters a morphological feature of a shoot that is regenerated from thetransformed cells, (b) regenerating shoots from the cells, (c)identifying shoots, or plants obtained therefrom, that (i) do not havean altered morphological feature compared to shoots or plantsregenerated from non-transformed cells, but (ii) do have the desiredpolynucleotide integrated into their genomes, wherein expression of thedesired polynucleotide modifies a trait in the shoot or plant.
 2. Themethod of claim 1, wherein the morphological feature that is altered bythe integration marker gene is at least any one of the shape, texure, orcolor of any one or more of the transformed shoot's leaves, roots, stem,or height
 3. The method of claim 1, wherein the integration marker geneis a cytokinin gene.
 4. The method of claim 3, wherein the cytokiningene is isopentenyl transferase, a transzeatine synthase, or a recessiveHoc1 gene.
 5. The method of claim 1, wherein the desired polynucleotideis located within a T-DNA or within a P-DNA that is positioned withinthe vector and the integration marker is not.
 6. The method of claim 5,wherein the desired polynucleotide consists essentially of nucleotidesequences that are native to the plant.
 7. A transformation vector,comprising a cytokinin gene in the backbone outside of a transformationcassette located in the vector.
 8. The transformation vector of claim 7,wherein the cytokinin gene is isolated from a bacterium or a plant. 9.The transformation vector of claim 7, wherein the cytokinin gene isselected from the group consisting of an isopentenyl transferase, atranszeatine synthase and a recessive Hoc1.
 10. The transformationvector of claim 7, wherein the transformation cassette is a T-DNA or aP-DNA.
 11. A method of transforming a plant cell with a desiredpolynucleotide, comprising infecting a plant cell with an Agrobacteriumstrain carrying a vector that comprises (i) a desired polynucleotide,(ii) a selectable marker gene, and (iii) a cytokinin gene located in thebackbone of the vector, wherein a cell that comprises the desiredpolynucleotide but not the integration marker gene in its genome is atransformed plant cell.
 12. The method of claim 11, further comprisingidentifying shoots regenerated from the infected plant cells thatdisplay a cytokinin-induced phenotype.
 13. The method of claim 11,wherein the cytokinin gene is selected from the group consisting of anisopentenyl transferase, a transzeatine synthase and a recessive Hoc1.14. The method of claim 12, further comprising growing plants fromshoots that do not display a cytokinin-induced phenotype.
 15. The methodof claim 11, wherein the vector further comprises a negative selectablemarker gene.
 16. The method of claim 15, wherein the negative selectablemarker gene is cytosine deaminase.
 17. The method of claim 11, whereinthe selectable marker gene is kanamycin or neomycin phosphotransferase.18. The method of claim 15, wherein the selectable marker gene and thenegative selectable marker gene are located on two differenttransformation vectors.